Compound or salt thereof having affinity substance for antibody, cleavable part and reactive group

Abstract

A compound or a salt thereof having an affinity substance for an antibody, a cleavable portion and a reactive group, wherein the compound is represented by the following formula (I): A-L-B-R (I) wherein A is an affinity substance for an antibody; L is a cleavable linker, which is a divalent group comprising a cleavable portion; B is (a) a divalent group comprising a bioorthogonal functional group or (b) a divalent group not containing a bioorthogonal functional group; and R is a reactive group for the antibody. Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus.

Description

Technical Field

[0001] The present invention relates to a compound having an affinity substance for an antibody, a cleavable moiety, and a reactive group, or a salt thereof. Background Art

[0002] In recent years, a large amount of research and development has been conducted on antibody-drug conjugates (ADCs). As the name suggests, ADCs are drugs (e.g., anticancer agents) conjugated to antibodies, and they have direct cytotoxic activity against cancer cells. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), jointly developed by Immunogene and Roche (Non-Patent Documents 1-3).

[0003] ADCs, such as T-DM1, have faced heterogeneity issues since their early stages of development. Specifically, because low-molecular-weight drugs are randomly reacted with the approximately 70 to 80 Lys residues present in antibodies, the drug-antibody ratio (DAR) and conjugation position are uncertain. It is generally known that with such random conjugation methods, the DAR ranges from 0 to 8, resulting in a variety of agents with varying numbers of drug conjugates. In recent years, reports have shown that altering the number and position of drug conjugates in an ADC can alter in vivo kinetics, drug release rate, and efficacy. Based on these findings, control over the number and position of conjugated drugs is required for next-generation ADCs. It is generally believed that by defining the number and position of conjugated drugs, the expected efficacy, diversity of conjugated agents, and batch variability, a so-called standardization issue, can be resolved (Non-Patent Document 4).

[0004] Research is underway globally on methods for regioselectively modifying antibodies, but nearly all involve genetic engineering or enzyme-based modification. While genetic engineering methods can control positional and population selectivity, they have been cited as problematic, such as reduced antibody expression efficiency (reduced overall yield during ADC production). Furthermore, the time required to construct antibody expression systems is a significant challenge (Non-Patent Documents 5-7).

[0005] Furthermore, in recent years, methods have been reported for chemically modifying proteins using small molecule probes in the complex environment of cells. This method is being used for receptor identification, for example, in imaging and the relocalization of low-molecular-weight drugs. Furthermore, in the field of biochemistry, organic chemical protein modification methods using synthetic small molecule probes are attracting attention (Non-Patent Documents 8-10).

[0006] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed. This method successfully achieves position-selective modification of antibodies by reacting a peptide reagent containing an NHS active ester and a drug linked to an affinity peptide (i.e., a method for preparing an ADC via a linker containing a peptide moiety). This method is the first in the world to successfully perform position-selective modification of the Fc region of an antibody using a drug through chemical synthesis, and has also demonstrated excellent results in actual use (reaction time 30 minutes, yield 70% (in the case of DAR 1), and position selectivity 100%). It has been demonstrated that the DAR can be controlled to 2 by adding approximately 5 equivalents of the peptide reagent, and that the modification position can also be controlled, which is groundbreaking (Patent Document 1).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2016 / 186206.

[0010] Non-patent literature

[0011] Non-patent document 1: Reichert JM et al., Nat Biotechnol 2005; 23: 1073-8

[0012] Non-patent document 2: Kubota T et al., Cancer Sci 2009; 100: 1566-72

[0013] Non-patent document 3: Wu AM et al., Nat Biotechnol 2005; 23: 1137-46

[0014] Non-patent document 4: Junutula JR et al., Nat Biotechnol 2008; 26: 925-32

[0015] Non-patent literature 5: Shen BQ et al., Nat Biotechnol 2012; 30: 184-9

[0016] Non-patent document 6: Hofer T et al., Biochemistry 2009; 48: 12047-57

[0017] Non-patent document 7: Liu W et al., Nat Methods 2007; 4: 239-44

[0018] Non-patent document 8: STLaughlin et al., Science 2008; 320,664

[0019] Non-patent literature 9: AE Spears et al., ChemBioChem 2004; 5, 41

[0020] Non-patent document 10: Y. Takaoka et al., Angew. Chem. Int. Ed. 2013; 52, 4088. Summary of the Invention

[0021] Technical problem to be solved by the invention

[0022] The object of the present invention is to develop a technology that enables modification of antibodies, particularly position-selective modification of antibodies.

[0023] Technical solutions used to solve technical problems

[0024] The present inventors have conducted intensive studies and, as a result, have discovered that a compound developed based on an original new design concept has the following structural features: (1) an affinity substance for an antibody (a substance having affinity for an antibody), (2) a reactive group for an amino acid residue constituting the antibody, (3) a cleavable portion between the affinity substance and the reactive group, and (4) a structural unit having a bioorthogonal functional group on the reactive group side (i.e., a structural unit comprising a bioorthogonal functional group and a reactive group) that can be generated by cleavage at the cleavable portion. The compound can be used for position-specific modification of antibodies (e.g. Figure 1-1 、 Figure 1-2 、 Figure 1-3 、 Figure 2 ).

[0025] The present inventors have also discovered that by using such compounds, antibodies (e.g., antibody-drug conjugates (ADCs)) can be prepared that do not contain a peptide portion as a linker and that positionally selectively contain a functional substance (e.g., a drug). In the clinical application of ADCs, it is ideal to avoid the use of linkers that are potentially immunogenic and contain a peptide portion that is easily hydrolyzed in the blood. That is, the method developed by the present inventors is the first in the world to successfully perform position-selective modification of the Fc region of an antibody with a drug by chemical synthesis means without using a linker containing a peptide portion. If such a method is adopted, it becomes possible to develop various compounds having structural characteristics such as those described above (1) to (4) (e.g., Figure 1-1 、 Figure 1-2 、 Figure 1-3 、 Figure 2 ).

[0026] Furthermore, the present inventors have discovered that by using a polypeptide containing a glutamine residue (Q) at the N-terminus as an affinity substance for antibodies, the amino group (-NH2) in the glutamine residue (Q) at the N-terminus can be protected by reacting the amino group (-NH2) with an amide group present in its side chain (for example, -CONH2 in the glutamine residue (Q) when the N-terminus contains QET) (pyroglutamylation). This allows a linker that mediates the binding of the affinity substance for antibodies to be specifically added to a specific amino acid residue (lysine residue having an amino group) in the affinity substance. Consequently, polypeptides containing a glutamine residue (Q) at the N-terminus are useful as affinity substances for antibodies.

[0027] Furthermore, the present inventors have discovered that a secretory production method for polypeptides using coryneform bacteria as hosts (International Publication No. 2013 / 062029) is useful for conveniently and massively producing polypeptides containing a glutamine residue (Q) at their N-termini. The present invention, by adding the N-terminal three residues Gln-Glu-Thr (QET) of the mature Csp protein to the N-terminus of the target polypeptide, can achieve the advantages of this secretory production method for polypeptides, making it suitable for producing polypeptides containing a glutamine residue (Q) at their N-termini (antibody-binding substances).

[0028] That is, the present invention is as follows.

[0029] In a first embodiment, the present invention provides a compound or a salt thereof having an affinity substance for an antibody, a cleavable portion, and a reactive group, and a reagent for position-selective modification of an antibody comprising the compound or the salt thereof;

[0030] [1] A compound having an affinity substance for an antibody, a cleavable moiety, and a reactive group, or a salt thereof, wherein the compound is represented by the following formula (I):

[0031] ALBR (I)

[0032] Where,

[0033] A is an affinity substance for antibodies,

[0034] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0035] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0036] R is a reactive group for the antibody (a group reactive with the antibody);

[0037] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0038] [2] The compound or salt thereof according to [1], wherein the polypeptide comprises a tripeptide (QET) consisting of a glutamine residue (Q)-glutamic acid (E)-threonine residue (T) at its N-terminus;

[0039] [3] The compound or salt thereof according to [1] or [2], wherein the glutamine residue is pyroglutamylated;

[0040] [4] The compound or salt thereof according to any one of [1] to [3], wherein the polypeptide has 16 or more and 123 or less amino acid residues;

[0041] [5] The compound or salt thereof according to any one of [1] to [3], wherein the polypeptide has 20 or more and 70 or less amino acid residues;

[0042] [6] The compound or salt thereof according to any one of [1] to [5], wherein the polypeptide is selected from the following (1) to (4):

[0043] [Chemical Formula 1]

[0044]

[0045] Q is the N-terminal glutamine residue, E is a glutamic acid residue, T is a threonine residue, the solid line is a bond, the spacer portion is a portion consisting of 1 or more and 50 or less amino acid residues, and the affinity substance for the antibody is a portion consisting of 15 or more and 70 or less amino acid residues;

[0046] [7] The compound or salt thereof according to [6], wherein the polypeptide portion having affinity for antibodies is selected from the group consisting of protein A, protein G, protein L, protein Z, fragments thereof having affinity for antibodies, and mutants thereof having affinity for antibodies;

[0047] [8] The compound or salt thereof according to [6] or [7], wherein the affinity polypeptide portion for the antibody comprises: (a) an amino acid sequence of FNMQCQR RFYEALHDPNLNEEQRNAXIXSIRDDC (SEQ ID NO: 5) (wherein each of the two Xs is independently an amino acid residue selected from an arginine residue, a glutamic acid residue, and a glutamine residue), wherein any one of the amino acid residues is substituted with a lysine residue, and the amino acid sequence has 90% or greater identity with the amino acid sequence of SEQ ID NO: 5;

[0048] [9] The compound or salt thereof according to any one of [6] to [8], wherein the affinity polypeptide portion for the antibody comprises: (a) an amino acid sequence in which any one of the amino acid residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 6) is substituted with a lysine residue, and the amino acid sequence has 90% or greater identity with the amino acid sequence of SEQ ID NO: 6;

[0049]

[10] The compound or salt thereof according to any one of [1] to [9], wherein the affinity substance for the antibody comprises an amino acid sequence selected from the group consisting of (1) to (11):

[0050] (1)QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7);

[0051] (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQID NO: 8);

[0052] (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9);

[0053] (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10);

[0054] (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22);

[0055] (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23);

[0056] (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51);

[0057] (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52);

[0058] (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53);

[0059] (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and

[0060] (11)QETRGNCAYHKGQIIWCTYH (SEQ ID NO:25);

[0061]

[11] The compound or salt thereof according to any one of [1] to

[10] , wherein L is:

[0062] (i) a cleavable linker, which is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group by cleavage; or

[0063] (ii) a cleavable linker, which is a divalent group comprising a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side by cleavage;

[0064]

[12] The compound or salt thereof according to

[11] , wherein L is the cleavable linker of (i);

[0065]

[13] The compound or salt thereof according to

[11] or

[12] , wherein L is the cleavable linker of (i), and B is the divalent group of (b);

[0066]

[14] The compound or salt thereof according to

[11] , wherein L is the cleavable linker of (ii), and B is the divalent group of (a);

[0067]

[15] The compound or salt thereof according to any one of [1] to

[14] , wherein the polypeptide is a peptide that binds to the Fc region of a monoclonal antibody;

[0068]

[16] The compound or salt thereof according to

[15] , wherein the polypeptide is a peptide that binds to the Fc region of IgG;

[0069]

[17] The compound or salt thereof according to any one of [1] to

[16] , wherein the affinity substance is an affinity substance for an antibody comprising an Fc region protein selected from any one of the following (A) to (C) and having antigen-binding ability:

[0070] (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;

[0071] (B) an Fc region protein comprising an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted into the amino acid sequence of SEQ ID NO: 1; or

[0072] (C) an Fc region protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1;

[0073]

[18] The compound or salt thereof according to any one of [1] to

[17] , wherein the polypeptide can bind to human IgG;

[0074]

[19] The compound or salt thereof according to any one of [1] to

[18] , wherein the cleavable portion is a portion that can be cleaved by any one of the following treatments (a) to (c),

[0075] (a) treatment with one or more substances selected from the group consisting of acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes;

[0076] (b) treatment with a physicochemical stimulus selected from light; or

[0077] (c) placement using a cleavable linker comprising an autolytically degradable cleavable moiety;

[0078]

[20] The compound or salt thereof according to any one of [1] to

[19] , wherein the cleavable moiety is selected from a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue;

[0079]

[21] The compound or salt thereof according to any one of

[11] to

[20] , wherein the cleavable moiety of (i) is selected from a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue;

[0080]

[22] The compound or salt thereof according to any one of

[11] to

[20] , wherein the cleavable moiety of (ii) is selected from an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue;

[0081]

[23] The compound or salt thereof according to any one of [1] to

[22] , wherein the cleavable moiety corresponds to any chemical structure selected from the following group;

[0082] [Chemical Formula 2]

[0083]

[0084] Here, the wavy lines perpendicular to the bonds indicate the cut sites.

[0085] Multiple R 2a , multiple R 2b and multiple R 2c Same or different, selected from

[0086] (i) a hydrogen atom or a halogen atom,

[0087] (ii) a monovalent hydrocarbon group,

[0088] (iii) an aralkyl group,

[0089] (iv) a monovalent heterocyclic group,

[0090] (v)R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or a monovalent hydrocarbon group,

[0091] (vi)NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group, or (vii) a nitro group, a sulfate group, a sulfonic acid group, a cyano group or a carboxyl group,

[0092] J is -CH2-, -O- or -S-,

[0093] r is an arbitrary integer from 1 to 4,

[0094] ○ (white circle) represents the bond with A, ● (black circle) represents the bond with B,

[0095] When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A, and ○ may represent the bond to B;

[0096]

[24] The compound or salt thereof according to any one of

[11] to

[19] ,

[21] , and

[23] , wherein the cleavable moiety of (i) corresponds to any chemical structure selected from the following group:

[0097] [Chemical Formula 3]

[0098]

[0099] Here, the wavy lines perpendicular to the bonds indicate the cut sites.

[0100] R 2a The meanings of the corresponding symbols are the same as those in

[23] .

[0101] ○ (white circle) represents the bond with A, ● (black circle) represents the bond with B,

[0102] When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A, and ○ may represent the bond to B;

[0103]

[25] The compound or salt thereof according to any one of

[11] to

[19] ,

[22] , and

[23] , wherein the cleavable moiety of (ii) corresponds to any chemical structure selected from the following group:

[0104] [Chemical Formula 4]

[0105]

[0106] Here, the wavy lines perpendicular to the bonds indicate the cut sites.

[0107] R 2b 、R 2c , J, r have the same meanings as the corresponding symbols in

[23] .

[0108] ○ (white circle) represents the bond with A, ● (black circle) represents the bond with B,

[0109] When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A, and ○ may represent the bond to B;

[0110]

[26] The compound or salt thereof according to any one of [1] to

[25] , wherein L is represented by any one of the following formulae (L1) to (L3); La-C-Lb (L1)

[0111] La-C (L2)

[0112] C-Lb (L3)

[0113] Where,

[0114] La and Lb are divalent groups,

[0115] C is the cutting part;

[0116]

[27] The compound or salt thereof according to

[26] , wherein La and Lb are represented by (La') and (Lb') respectively;

[0117] [Chemical Formula 5]

[0118]

[0119] Where,

[0120] p and p' are the same or different and are any integer from 0 to 10,

[0121] q and q' are the same or different and are any integer from 0 to 10,

[0122] X and X' are the same or different and are carbon atoms, nitrogen atoms or single bonds (where X is a nitrogen atom, R 1b does not exist; when X' is a nitrogen atom, R 1b' Does not exist. When X is a single bond, R 1a and R 1b Does not exist; when X' is a single bond, R 1a' and R 1b' does not exist),

[0123] R 1a 、R 1b 、R 1a' and R 1b' The same or different atoms or groups are selected from (i) to (vii);

[0124]

[28] The compound or salt thereof according to any one of [1] to

[27] , wherein the divalent group containing a bioorthogonal functional group is a divalent group containing a bioorthogonal functional group selected from the group consisting of an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an olefin residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halogenated carbonyl residue, an isonitrile residue, a sydnone residue, and a selenium residue in the main chain;

[0125]

[29] The compound or salt thereof according to any one of [1] to

[27] , wherein the divalent group containing a bioorthogonal functional group is a divalent group containing a bioorthogonal functional group selected from the group consisting of an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an olefin residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, an α-halogenated carbonyl residue, an isonitrile residue, a sterone residue, and a selenium residue in a side chain;

[0126]

[30] The compound or salt thereof according to any one of [1] to

[29] , wherein the bioorthogonal functional group is any one of the following formulae:

[0127] [Chemical Formula 6]

[0128]

[0129] Where,

[0130] R 1f , single or multiple R 1g , and single or multiple R 1h are the same or different and are atoms, groups or electron-withdrawing groups selected from (i) to (vii),

[0131] ·Indicates a bond;

[0132]

[31] The compound or salt thereof according to any one of [1] to

[30] , wherein the divalent group of (b) is selected from a substituted alkylene group, a substituted cycloalkylene group, a substituted aryl group, a substituted divalent heterocyclic group, -NR a -(R a represents a hydrogen atom or a substituent), -O-, or a combination of two or more of these groups;

[0133]

[32] The compound or salt thereof according to any one of [1] to

[31] , wherein B is represented by the following formula (B-1);

[0134] [Chemical Formula 7]

[0135]

[0136] In formula (B-1),

[0137] Y is -NH-, -O-, -CH2- or the following formula (B-2),

[0138] [Chemical Formula 8]

[0139]

[0140] (In formula (B-2),

[0141] V and V' are the same or different and are -NH-, -O-, -CH2- or a single bond,

[0142] V1 is a divalent group containing a bioorthogonal functional group,

[0143] s is an arbitrary integer from 0 to 10,

[0144] The orientations of ○ and ● in formula (B-2) are the same as those of ○ and ● in formula (B-1),

[0145] Z is an oxygen atom, a sulfur atom or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH2-),

[0146] In formula (B-1), ○ (white circle) represents a bond to the L-side moiety, and ● (black circle) represents a bond to the R-side moiety.

[0147]

[33] The compound or salt thereof according to any one of [1] to

[32] , wherein the reactive group is a reactive group specific for the side chain of any one of a lysine residue, a tyrosine residue, and a tryptophan residue;

[0148]

[34] The compound or salt thereof according to

[33] , wherein the reactive group is a reactive group specific for the side chain of a lysine residue;

[0149]

[35] The compound or salt thereof according to any one of [1] to

[34] , wherein the reactive group corresponds to any chemical structure selected from the following group;

[0150] [Chemical Formula 9]

[0151]

[0152] Here,

[0153] R 5a and R 5c is an atom or group selected from (i) to (vii),

[0154] R 5b is an electron-withdrawing group,

[0155] j is an arbitrary integer from 1 to 5,

[0156] k is any integer from 1 to 4;

[0157]

[36] The compound or salt thereof according to any one of [1] to

[35] , wherein the number of atoms in the main chain connecting A and R is 4 to 20;

[0158]

[37] The compound or salt thereof according to any one of [1] to

[36] , wherein the main chain connecting A and R does not contain a ring structure;

[0159]

[38] The compound or salt thereof according to any one of [1] to

[37] , wherein the partial structure represented by LB does not contain a peptide portion;

[0160]

[39] The compound or salt thereof according to any one of [1] to

[38] , wherein the compound represented by the formula (I) is a compound represented by the following formula (I');

[0161] A-B2-L'-B1-R (I')

[0162] Where,

[0163] A and R have the same meanings as the corresponding symbols in formula (I),

[0164] L' is a cleavable linker, which is a divalent group comprising a cleavable moiety, B1 and B2 are the same or different and are (a) a divalent group comprising a bioorthogonal functional group, or (b) a divalent group not comprising a bioorthogonal functional group,

[0165] B1 and B2 may have a symmetrical structure centered on L';

[0166]

[40] The compound or salt thereof according to

[39] , wherein the compound represented by the formula (I') is represented by the following formula (I'');

[0167] [Chemical Formula 10]

[0168]

[0169] Where,

[0170] A and R have the same meanings as the corresponding symbols in formula (I) described in [1],

[0171] C is the cutting part,

[0172] p, p', q, q', X, X', R 1a 、R 1a' 、R 1b and R 1b' The meanings of the corresponding symbols in formula (La') and (Lb') described in

[19] are the same,

[0173] Y and Y' are the same or different and are the same as Y in formula (B-1) described in

[32] ,

[0174] Z and Z' are the same or different and are the same as Z in formula (B-1);

[0175]

[41] A reagent for position-selective modification of an antibody (preferably comprising the compound or a salt thereof according to any one of [2] to

[40] above), wherein the compound is represented by the following formula (I) and comprises a substance having affinity for the antibody, a cleavable moiety, and a reactive group, or a salt thereof;

[0176] ALBR (I)

[0177] Where,

[0178] A is an affinity substance for antibodies,

[0179] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0180] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0181] R is a reactive group against the antibody;

[0182] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0183]

[42] A compound having an affinity substance for an antibody, a cleavable portion and a reactive group, or a salt thereof, wherein the compound is represented by the following formula (I):

[0184] ALBR (I)

[0185] Where,

[0186] A is an affinity substance for antibodies,

[0187] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0188] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0189] R is a reactive group specific for the side chain of a lysine residue;

[0190] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0191]

[43] A position-selective modification reagent for an antibody, wherein:

[0192] A compound represented by the following formula (I) and comprising a substance having affinity for an antibody, a cleavable portion and a reactive group, or a salt thereof,

[0193] ALBR (I)

[0194] Where,

[0195] A is an affinity substance for antibodies,

[0196] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0197] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0198] R is a reactive group specific for the side chain of a lysine residue;

[0199] Furthermore, the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0200] In a second aspect, the present invention provides an antibody or a salt thereof having an affinity substance for the antibody and a cleavage portion, and a method for producing the same;

[0201] (Antibody or its salt having an affinity substance for the antibody and a cleavable portion)

[0202] [1] An antibody or a salt thereof having an affinity substance for the antibody and a cleavable portion, wherein:

[0203] It is represented by the following formula (II),

[0204] ALB-R'-T (II)

[0205] Where,

[0206] A is an affinity substance for antibodies,

[0207] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0208] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0209] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0210] T is antibody;

[0211] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0212] [2] The antibody or salt thereof according to [1], wherein the antibody is a monoclonal antibody;

[0213] [3] The antibody or salt thereof according to [1] or [2], wherein the antibody is an IgG antibody;

[0214] [4] The antibody or salt thereof according to any one of [1] to [3], wherein the antibody is of human origin;

[0215] [5] The antibody or salt thereof according to any one of [1] to [4], wherein the antibody comprises an Fc region protein selected from any one of the following (A) to (C) and has antigen-binding ability:

[0216] (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;

[0217] (B) an Fc region protein comprising an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted into the amino acid sequence of SEQ ID NO: 1; or

[0218] (C) an Fc region protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1;

[0219] [6] The antibody or a salt thereof according to any one of [1] to [5], wherein the antibody contains one or more specific amino acid residues in a target region formed by 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region,

[0220] The structural unit represented by ALB-R' binds to one or more specific amino acid residues contained in the target region with a position selectivity of more than 30%;

[0221] [7] The antibody or salt thereof according to [6], wherein the target region is a region formed by 1 to 10 consecutive amino acid residues;

[0222] [8] The antibody or salt thereof according to [7], wherein the target region is a region formed by 1 to 3 consecutive amino acid residues;

[0223] [9] The antibody or salt thereof according to [8], wherein the target region is a region formed by amino acid residues 246 to 248 in the human IgG Fc region;

[0224]

[10] The antibody or salt thereof according to any one of [6] to [9], wherein the position selectivity is 50% or more;

[0225]

[11] The antibody or salt thereof according to

[10] , wherein the position selectivity is 70% or more;

[0226]

[12] The antibody or salt thereof according to

[10] , wherein the position selectivity is 90% or more;

[0227]

[13] The antibody or salt thereof according to any one of [6] to

[12] , wherein the specific amino acid residue, when centered on the specific amino acid at the specific position, does not contain any amino acid residue of the same type as the specific amino acid residue in the region extending from the N-terminal side and the C-terminal side to a position separated by a amino acid residues, other than the specific amino acid residue at the specific position; wherein a is an arbitrary integer from 1 to 10;

[0228]

[14] The antibody or salt thereof according to any one of [1] to

[13] , wherein the antibody comprises multiple heavy chains, T has a structural unit represented by ALB-R' in corresponding multiple target regions in the multiple heavy chains, and thus the antibody has multiple structural units represented by ALB-R';

[0229]

[15] The antibody or salt thereof according to

[14] , wherein the number of heavy chains is 2;

[0230]

[16] The antibody or salt thereof according to any one of [1] to

[15] , wherein the portion produced by the reaction between the antibody and the reactive group is a portion produced by the reaction between a lysine residue, a tyrosine residue, or a tryptophan residue and a reactive group specific for the side chain of any one of the lysine residue, the tyrosine residue, and the tryptophan residue;

[0231]

[17] The antibody or salt thereof according to any one of [1] to

[16] , wherein the portion produced by the reaction between the antibody and the reactive group is: a portion produced by the reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue;

[0232]

[18] The antibody or salt thereof according to any one of [1] to

[17] , wherein the portion generated by the reaction corresponds to any chemical structure selected from the following group;

[0233] [Chemical Formula 11]

[0234]

[0235] Here, ● (black circle) represents a bond bound to the T-side portion, ○ (white circle) represents a bond bound to the B-side portion, and a straight line perpendicular to the bond represents a bond generated by the reaction;

[0236]

[19] The antibody or salt thereof according to any one of [1] to

[18] , wherein the number of atoms in the main chain linking A and R' is 4 to 20;

[20] The antibody or salt thereof according to any one of [1] to

[19] , wherein the main chain linking A and R does not contain a ring structure;

[0237]

[21] The antibody or salt thereof according to any one of [1] to

[20] , wherein the partial structure represented by LB does not contain a peptide portion;

[0238]

[22] The antibody or salt thereof according to any one of [1] to

[21] , wherein the compound represented by the formula (II) is a compound represented by the following formula (II');

[0239] A-B2-L'-B1-R'-T (II')

[0240] Where,

[0241] A, R' and T have the same meanings as the corresponding symbols in formula (II),

[0242] L' is a cleavable linker, which is a divalent group comprising a cleavable moiety, B1 and B2 are the same or different and are (a) a divalent group comprising a bioorthogonal functional group, or (b) a divalent group not comprising a bioorthogonal functional group,

[0243] B1 and B2 may have a symmetrical structure centered on L';

[0244]

[23] The antibody or salt thereof according to

[22] , wherein the compound represented by the formula (II') is represented by the following formula (II");

[0245] [Chemical Formula 12]

[0246]

[0247] Where,

[0248] A, R' and T have the same meanings as the corresponding symbols in the above formula (II),

[0249] C is the cutting part,

[0250] p and p' are the same or different and are any integer from 0 to 10,

[0251] q and q' are the same or different and are any integer from 0 to 10,

[0252] X and X' are the same or different and are carbon atoms, nitrogen atoms or single bonds (where X is a nitrogen atom, R 1b does not exist; when X' is a nitrogen atom, R 1b' Does not exist. When X is a single bond, R 1a and R 1b Does not exist; when X' is a single bond, R 1a' and R 1b' does not exist),

[0253] R 1a、R 1b 、R 1a' and R 1b' the same or different, selected from (i) a hydrogen atom or a halogen atom,

[0254] (ii) a monovalent hydrocarbon group,

[0255] (iii) an aralkyl group,

[0256] (iv) a monovalent heterocyclic group,

[0257] (v)R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or a monovalent hydrocarbon group,

[0258] (vi)NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group, or (vii) a nitro group, a sulfate group, a sulfonic acid group, a cyano group or a carboxyl group,

[0259] Y and Y' are the same or different and are the same as Y in the above formula (B-1),

[0260] Z and Z' are the same or different and are the same as Z in formula (B-1);

[0261]

[24] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, wherein:

[0262] It is represented by the following formula (II),

[0263] ALB-R'-T (II)

[0264] Where,

[0265] A is an affinity substance for antibodies,

[0266] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0267] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R' is a moiety generated by the reaction between the antibody and a reactive group specific for the side chain of a lysine residue, T is the antibody,

[0268] Furthermore, the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0269] (Method for producing an antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion)

[0270]

[25] A method for producing an antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, comprising:

[0271] ALBR (I)

[0272] The compound or its salt having an affinity substance for the antibody, a cleavable portion and a reactive group represented by the formula (II) is reacted with the antibody to generate the following compound:

[0273] ALB-R'-T (II)

[0274] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion,

[0275] In formula (I),

[0276] A is an affinity substance for antibodies,

[0277] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0278] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0279] R is a reactive group against the antibody;

[0280] In formula (II),

[0281] A, L and B have the same meanings as the corresponding symbols in formula (I),

[0282] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0283] T is antibody;

[0284] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0285]

[26] The method according to

[25] , wherein the reactive group is a reactive group specific for the side chain of a lysine residue.

[0286] In a third embodiment, the present invention provides a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, and a method for producing the same;

[0287] (Complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof)

[0288] [1] A complex comprising a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, wherein:

[0289] The complex is represented by the following formula (III):

[0290] AL-B'(-F)-R'-T (III)

[0291] Where,

[0292] A is an affinity substance for antibodies,

[0293] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0294] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0295] F is a functional substance,

[0296] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0297] T is antibody,

[0298] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0299] [2] The complex or a salt thereof according to [1], wherein the antibody is a monoclonal antibody;

[0300] [3] The complex or salt thereof according to [1] or [2], wherein the antibody is an IgG antibody;

[0301] [4] The complex or salt thereof according to any one of [1] to [3], wherein the antibody is derived from human;

[0302] [5] The complex or salt thereof according to any one of [1] to [4], wherein the antibody is an antibody comprising any one of the Fc region proteins selected from the following (A) to (C) and having antigen-binding ability:

[0303] (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;

[0304] (B) an Fc region protein comprising an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted into the amino acid sequence of SEQ ID NO: 1; or

[0305] (C) an Fc region protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1;

[0306] [6] The complex or salt thereof according to any one of [1] to [5], wherein the antibody contains one or more specific amino acid residues in a target region formed by 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region,

[0307] The structural unit represented by AL-B'(-F)-R' binds to one or more specific amino acid residues contained in the target region with a position selectivity of more than 30%;

[0308] [7] The complex or a salt thereof according to [6], wherein the specific amino acid residue, when centered on the specific amino acid at the specific position, does not contain any amino acid residue of the same type as the specific amino acid residue in the region extending from the N-terminal side and the C-terminal side to a position separated by a amino acid residues, other than the specific amino acid residue at the specific position; wherein a is an arbitrary integer from 1 to 10;

[0309] [8] The complex or salt thereof according to any one of [1] to [7], wherein the antibody is an antibody comprising multiple heavy chains, T has a structural unit represented by AL-B'(-F)-R' in multiple target regions corresponding to the multiple heavy chains, and thus the antibody has multiple structural units represented by AL-B'(-F)-R';

[0310] [9] The complex or salt thereof according to any one of [1] to [8], wherein the functional substance has a functional group that readily reacts with a bioorthogonal functional group or is derivatized so as to have a functional group that readily reacts with a bioorthogonal functional group, and the functional group that readily reacts with a bioorthogonal functional group is a group selected from an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an olefin residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halogenated carbonyl residue, an isonitrile residue, a sterone residue, and a selenium residue;

[0311]

[10] The complex or salt thereof according to any one of [1] to [9], wherein the functional substance has a functional group that readily reacts with a bioorthogonal functional group or is derivatized so as to have a functional group that readily reacts with a bioorthogonal functional group, and the functional group that readily reacts with a bioorthogonal functional group is a group selected from the following groups:

[0312] [Chemical Formula 13]

[0313]

[0314] Where,

[0315] R 1f , single or multiple R 1g and single or multiple R 1h are the same or different, are atoms or groups selected from (i) to (vii), or electron-withdrawing groups, and are bonds that bind to functional substances;

[0316]

[11] The complex or salt thereof according to any one of [1] to

[10] , wherein the portion produced by the reaction between the antibody and the reactive group is a portion produced by the reaction between a lysine residue, a tyrosine residue, or a tryptophan residue and a reactive group specific for the side chain of any one of the lysine residue, the tyrosine residue, and the tryptophan residue;

[0317]

[12] The complex or salt thereof according to any one of [1] to

[11] , wherein the portion produced by the reaction between the antibody and the reactive group is: a portion produced by the reaction between a lysine residue and a reactive group specific for the side chain of the lysine residue;

[0318]

[13] The complex or salt thereof according to any one of [1] to

[12] , wherein the portion generated by the reaction corresponds to any chemical structure selected from the following group;

[0319] [Chemical Formula 14]

[0320]

[0321] Here, ● (black circle) represents a bond bound to the moiety on the T side, and ○ (white circle) represents a bond bound to the moiety on the B side;

[0322]

[14] The complex or salt thereof according to any one of [1] to

[13] , wherein the functional substance is a drug or a labeling substance;

[0323]

[15] The complex or salt thereof according to any one of [1] to

[13] , wherein the functional substance is a low molecular weight compound;

[0324]

[16] The complex or salt thereof according to

[14] or

[15] , wherein the drug is an anticancer agent;

[0325]

[17] The complex or salt thereof according to any one of [1] to

[16] , wherein the number of atoms in the main chain connecting A and R' is 4 to 20;

[0326]

[18] The complex or salt thereof according to any one of [1] to

[17] , wherein the main chain connecting A and R does not contain a ring structure;

[0327]

[19] The complex or salt thereof according to any one of [1] to

[18] , wherein the partial structure represented by LB does not contain a peptide portion;

[0328]

[20] The complex or salt thereof according to any one of [1] to

[19] , wherein the compound represented by the formula (III) is represented by the following formula (III');

[0329] A-B2'(-F2)-L'-B1'(-F1)-R'-T (III')

[0330] Where,

[0331] A, R' and T have the same meanings as the corresponding symbols in formula (II),

[0332] L' is a cleavable linker, which is a divalent group containing a cleavable portion, B1' and B2' are the same or different and are a trivalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0333] F1 and F2 are the same or different and are functional substances.

[0334] B1'(-F1) and B2'(-F2) may have a symmetrical structure centered on L';

[0335]

[21] The complex or salt thereof according to

[20] , wherein the compound represented by the formula (III') is represented by the following formula (III");

[0336] [Chemical Formula 15]

[0337]

[0338] Where,

[0339] A, R' and T have the same meanings as the corresponding symbols in the above formula (III),

[0340] C is the cutting part,

[0341] p and p' are the same or different and are any integer from 0 to 10,

[0342] q and q' are the same or different and are any integer from 0 to 10,

[0343] X and X' are the same or different and are carbon atoms, nitrogen atoms or single bonds (where X is a nitrogen atom, R 1b does not exist; when X' is a nitrogen atom, R 1b' Does not exist. When X is a single bond, R 1a and R 1b Does not exist; when X' is a single bond, R 1a' and R 1b' does not exist),

[0344] R 1a 、R 1b 、R 1a' and R 1b' The same or different, selected from (i) to (vii),

[0345] Y and Y' are the same or different and are residues obtained by removing one hydrogen atom from Y of the above formula (B-1),

[0346] Z and Z' are the same or different and are the same as Z in formula (B-1),

[0347] F and F' are the same or different and are functional substances;

[0348]

[22] A complex comprising an affinity substance, a functional substance and an antibody, or a salt thereof, wherein:

[0349] The complex is represented by the following formula (III):

[0350] AL-B'(-F)-R'-T (III)

[0351] Where,

[0352] A is an affinity substance for antibodies,

[0353] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0354] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0355] F is a functional substance,

[0356] R' is a moiety generated by the reaction between the antibody and a reactive group specific for the side chain of a lysine residue, T is the antibody,

[0357] Furthermore, the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0358] (Method for producing a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof)

[0359]

[23] A method for producing a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, comprising:

[0360] With the following formula (II):

[0361] ALB-R'-T (II)

[0362] The antibody or its salt having an affinity substance for the antibody and a cleavable portion represented by is reacted with a functional substance to generate the following formula (III):

[0363] AL-B'(-F)-R'-T (III)

[0364] A complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody, or a salt thereof, wherein:

[0365] A is an affinity substance for antibodies,

[0366] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0367] B is (a) a divalent group containing a bioorthogonal functional group,

[0368] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0369] T is antibody;

[0370] In formula (III),

[0371] A, L, R' and T have the same meanings as the corresponding symbols in formula (II),

[0372] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0373] F is a functional substance;

[0374] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0375]

[24] The method according to

[23] , wherein the reactive group is a reactive group specific for the side chain of a lysine residue;

[0376]

[25] A method for producing a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, comprising:

[0377] (A) using the following formula (I):

[0378] ALBR (I)

[0379] The compound or its salt reacts with the antibody,

[0380] The following formula (II) is generated:

[0381] ALB-R'-T (II)

[0382] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion,

[0383] In formula (I),

[0384] A is an affinity substance for antibodies,

[0385] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0386] B is (a) a divalent group containing a bioorthogonal functional group,

[0387] R is a reactive group to the antibody,

[0388] In formula (II),

[0389] A, L and B have the same meanings as the corresponding symbols in formula (I),

[0390] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0391] T is antibody;

[0392] as well as

[0393] (B) reacting the antibody or its salt having an affinity substance for the antibody and a cleavable portion with a functional substance to generate a compound of the following formula (III):

[0394] AL-B'(-F)-R'-T (III)

[0395] A complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody, or a salt thereof, wherein:

[0396] A and L have the same meanings as the corresponding symbols in formula (I),

[0397] R' and T have the same meanings as the corresponding symbols in formula (II),

[0398] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0399] F is a functional substance;

[0400] Furthermore, the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0401] In a fourth embodiment, the present invention provides a method for producing an antibody having a bioorthogonal functional group;

[0402] [1] A method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising:

[0403] With the following formula (II):

[0404] ALB-R'-T (II)

[0405] The antibody or its salt is cleaved by a cleavage moiety of an antibody or a salt thereof having an affinity substance for the antibody and a cleavage moiety to generate the following formula (IV):

[0406] L1-B-R'-T(IV)

[0407] An antibody or a salt thereof having a bioorthogonal functional group;

[0408] In formula (II),

[0409] A is an affinity substance for antibodies,

[0410] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0411] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0412] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0413] T is antibody;

[0414] In formula (IV),

[0415] B, R' and T have the same meanings as the corresponding symbols in formula (II),

[0416] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; and the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus;

[0417] [2] The method according to [1], wherein L is:

[0418] (i) a cleavable linker, which is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group by cleavage; or

[0419] (ii) a cleavable linker, which is a divalent group comprising a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side by cleavage;

[0420] [3] The method according to [2], wherein L is the cleavable linker of (i),

[0421] L1 is (i') a monovalent group containing a bioorthogonal functional group,

[0422] B is a divalent group of (a) or (b);

[0423] [4] The method according to [2] or [3], wherein

[0424] L is the cleavable linker of (i),

[0425] L1 is (i') a monovalent group containing a bioorthogonal functional group,

[0426] B is the divalent group of (b);

[0427] [5] The method according to [2], wherein

[0428] L is the cleavable linker of (ii),

[0429] L1 is (i') a monovalent group without a bioorthogonal functional group,

[0430] B is the divalent group of (a);

[0431] [6] The method according to any one of [1] to [5], wherein the reactive group is a reactive group specific for the side chain of a lysine residue;

[0432] [7] A method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising:

[0433] (A) using the following formula (I):

[0434] ALBR (I)

[0435] The compound or its salt reacts with the antibody,

[0436] The following formula (II) is generated:

[0437] ALB-R'-T (II)

[0438] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion,

[0439] In formula (I),

[0440] A is an affinity substance for antibodies,

[0441] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0442] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0443] R is a reactive group to the antibody,

[0444] In formula (II),

[0445] A, L and B have the same meanings as the corresponding symbols in formula (I),

[0446] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0447] T is antibody;

[0448] as well as

[0449] (B) cleaving the antibody or its salt having an affinity substance for the antibody and a cleavable portion to generate a compound of the following formula (IV):

[0450] L1-B-R'-T (IV)

[0451] An antibody or a salt thereof having a bioorthogonal functional group,

[0452] In formula (IV),

[0453] B, R' and T have the same meanings as the corresponding symbols in formula (II),

[0454] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; and the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0455] In a fifth embodiment, the present invention provides a method for producing an antibody or a salt thereof having a functional substance;

[0456] [1] A method for producing an antibody or a salt thereof having a functional substance, comprising:

[0457] With the following formula (III):

[0458] AL-B'(-F)-R'-T (III)

[0459] The substance having affinity for the antibody, the cleavage portion, the complex of the functional substance and the antibody or the cleavage portion of the salt thereof is cleaved,

[0460] Generate the following formula (V1):

[0461] L1-B'(-F)-R'-T (V1)

[0462] An antibody or a salt thereof having a functional substance represented by

[0463] In formula (III),

[0464] A is an affinity substance for antibodies,

[0465] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0466] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0467] F is a functional substance,

[0468] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0469] T is antibody;

[0470] In formula (V1),

[0471] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group,

[0472] B', F, R' and T have the same meanings as the corresponding symbols in formula (III);

[0473] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0474] [2] The method according to [1], wherein the reactive group is a reactive group specific for the side chain of a lysine residue;

[0475] [3] A method for producing an antibody or a salt thereof having a functional substance, comprising:

[0476] (A) With the following formula (II):

[0477] ALB-R'-T (II)

[0478] The antibody or its salt having an affinity substance for the antibody and a cleavable portion represented by is reacted with a functional substance to generate the following formula (III):

[0479] AL-B'(-F)-R'-T (III)

[0480] A complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody, or a salt thereof, wherein:

[0481] A is an affinity substance for antibodies,

[0482] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0483] B is (a) a divalent group containing a bioorthogonal functional group,

[0484] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0485] T is antibody;

[0486] In formula (III),

[0487] A, L, R' and T have the same meanings as the corresponding symbols in formula (II),

[0488] B' is a trivalent group including a portion generated by a reaction between a functional substance and a bioorthogonal functional group, and F is a functional substance;

[0489] as well as

[0490] (B) cleaving the cleavage portion of the complex of the substance having affinity for the antibody, the cleavage portion, the functional substance and the antibody, or a salt thereof,

[0491] Generate the following formula (V1):

[0492] L1-B'(-F)-R'-T (V1)

[0493] An antibody or a salt thereof having a functional substance represented by

[0494] In formula (V1),

[0495] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group,

[0496] B', F, R' and T have the same meanings as the corresponding symbols in formula (III);

[0497] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0498] [4] A method for producing an antibody or a salt thereof having a functional substance, comprising:

[0499] (A) using the following formula (I):

[0500] ALBR (I)

[0501] The compound or its salt having an affinity substance for the antibody, a cleavable portion and a reactive group represented by the formula (II) is reacted with the antibody to generate the following compound:

[0502] ALB-R'-T (II)

[0503] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion,

[0504] In formula (I),

[0505] A is an affinity substance for antibodies,

[0506] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0507] B is (a) a divalent group containing a bioorthogonal functional group,

[0508] R is a reactive group to the antibody,

[0509] In formula (II),

[0510] A, L and B have the same meanings as the corresponding symbols in formula (I),

[0511] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0512] T is antibody;

[0513] (B) reacting the antibody or its salt having an affinity substance for the antibody and a cleavable portion with a functional substance to generate a compound of the following formula (III):

[0514] AL-B'(-F)-R'-T (III)

[0515] A complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody, or a salt thereof, wherein:

[0516] A and L have the same meanings as the corresponding symbols in formula (I),

[0517] R' and T have the same meanings as the corresponding symbols in formula (II),

[0518] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0519] F is a functional substance;

[0520] as well as

[0521] (C) cleaving the cleavage portion of the complex of the substance having affinity for the antibody, the cleavage portion, the functional substance and the antibody, or a salt thereof,

[0522] Generate the following formula (V1):

[0523] L1-B'(-F)-R'-T (V1)

[0524] An antibody or a salt thereof having a functional substance represented by

[0525] In formula (V1),

[0526] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group, and B', F, R' and T have the same meanings as the corresponding symbols in formula (III);

[0527] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0528] [5] A method for producing an antibody or a salt thereof having a functional substance, comprising:

[0529] (A) is the following formula (II):

[0530] ALB-R'-T (II)

[0531] The antibody or its salt is cleaved by a cleavage moiety of an antibody or a salt thereof having an affinity substance for the antibody and a cleavage moiety to generate the following formula (IV):

[0532] L1-B-R'-T (IV)

[0533] An antibody or a salt thereof having a bioorthogonal functional group,

[0534] In formula (II),

[0535] A is an affinity substance for antibodies,

[0536] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0537] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0538] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0539] T is antibody;

[0540] In formula (IV),

[0541] B, R' and T have the same meanings as the corresponding symbols in formula (II),

[0542] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; and

[0543] (B) reacting the antibody or salt thereof having a bioorthogonal functional group with one or more functional substances to generate a compound of the following formula (V2):

[0544] F-L1'-B-R'-T (V2)

[0545] Or the following formula (V3):

[0546] Fa-L1'-B'(-Fb)-R'-T (V3)

[0547] An antibody or a salt thereof having a functional substance represented by

[0548] In formula (V2),

[0549] B, R' and T have the same meanings as the corresponding symbols in formula (IV),

[0550] L1′ is a divalent group comprising a portion generated by a reaction between a functional substance and (i′) a monovalent group comprising a bioorthogonal functional group,

[0551] F is a functional substance,

[0552] In formula (V3),

[0553] R' and T have the same meanings as the corresponding symbols in formula (IV),

[0554] L1' has the same meaning as the corresponding symbols in formula (V2),

[0555] B' is a divalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0556] Fa and Fb are the same or different functional substances;

[0557] Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus;

[0558] [6] A method for producing an antibody or a salt thereof having a functional substance, comprising:

[0559] (A) using the following formula (I):

[0560] ALBR (I)

[0561] The compound or its salt reacts with the antibody,

[0562] The following formula (II) is generated:

[0563] ALB-R'-T (II)

[0564] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion,

[0565] In formula (I),

[0566] A is an affinity substance for antibodies,

[0567] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0568] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0569] R is a reactive group to the antibody,

[0570] In formula (II),

[0571] A, L and B have the same meanings as the corresponding symbols in formula (I),

[0572] R' is a moiety generated by the reaction between the antibody and the reactive group,

[0573] T is antibody;

[0574] (B) cleaving the antibody or its salt having an affinity substance for the antibody and a cleavable portion to generate a compound of the following formula (IV):

[0575] L1-B-R'-T (IV)

[0576] An antibody or a salt thereof having a bioorthogonal functional group,

[0577] In formula (IV),

[0578] B, R' and T have the same meanings as the corresponding symbols in formula (II),

[0579] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; and

[0580] (C) reacting the antibody or salt thereof having a bioorthogonal functional group with one or more functional substances to generate a compound of the following formula (V2):

[0581] F-L1'-B-R'-T (V2)

[0582] Or the following formula (V3):

[0583] Fa-L1'-B'(-Fb)-R'-T (V3)

[0584] An antibody or a salt thereof having a functional substance represented by

[0585] In formula (V2),

[0586] B, R' and T have the same meanings as the corresponding symbols in formula (IV),

[0587] L1′ is a divalent group comprising a portion generated by a reaction between a functional substance and (i′) a monovalent group comprising a bioorthogonal functional group,

[0588] F is a functional substance,

[0589] In formula (V3),

[0590] R' and T have the same meanings as the corresponding symbols in formula (IV),

[0591] L1' has the same meaning as the corresponding symbols in formula (V2),

[0592] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0593] Fa and Fb are the same or different functional substances;

[0594] Furthermore, the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0595] Effects of the Invention

[0596] (I) The compound of the present invention or a salt thereof having an affinity substance for an antibody, a cleavable moiety, and a reactive group can be used, for example, for position-selective modification of an antibody;

[0597] (I) The compound of the present invention or its salt, (II) the antibody of the present invention or its salt having (position-selectively) an affinity substance for an antibody and a cleavage portion, (III) the complex of the present invention or its salt having (position-selectively) an affinity substance for an antibody, a cleavage portion, a functional substance, and an antibody, can be used as, for example, an intermediate for preparing an antibody or its salt having (position-selectively) a bioorthogonal functional group, and an antibody or its salt having (position-selectively) a functional substance. The antibody or its salt having (position-selectively) a functional substance can be used as, for example, a drug or a reagent (e.g., a diagnostic drug, a research reagent). The antibody or its salt having (position-selectively) a bioorthogonal functional group can be used as an intermediate for preparing an antibody or its salt having (position-selectively) a functional substance. Therefore, (I) the compound of the present invention or its salt, (II) the antibody or its salt of the present invention, and (III) the complex or its salt of the present invention can be used as, for example, a synthetic intermediate for a drug or a reagent;

[0598] Furthermore, by using a polypeptide containing a glutamine residue (Q) at the N-terminus as an affinity substance for an antibody, the amino group (-NH2) in the glutamine residue (Q) at the N-terminus can be protected by reacting the amino group (-NH2) with an amide group present in its side chain (e.g., -CONH2 in the glutamine residue (Q) when the N-terminus contains QET) (pyroglutamylation). This allows the linker that mediates the binding of the affinity substance to the antibody to be more specifically added to a specific amino acid residue in the affinity substance.

[0599] Furthermore, if a secretory production method for polypeptides using coryneform bacteria as a host is adopted (CspB fusion method), the advantages of the polypeptide secretory production method can be enjoyed by adding the N-terminal three residues Gln-Glu-Thr (QET) of the CspB mature protein to the N-terminus of the target polypeptide, and it is therefore suitable for the preparation of polypeptides containing a glutamine residue (Q) at the N-terminus (affinity substances for antibodies).

[0600] Brief description of the accompanying drawings

[0601] Figure 1-1This is a schematic diagram (part one) showing the concept (concept) of position-selective modification of an antibody using the compound of the present invention [a compound having an affinity substance for an antibody, a cleavage moiety, and a reactive group: ALBR (I)]. First, the compound of the present invention associates with the antibody (T) via the affinity substance (A) for the antibody. Next, the compound of the present invention reacts with the side chain of a specific amino acid residue (the side chain of a lysine residue in the figure) in the target region near the association site between the affinity substance and the antibody via the reactive group (R) (an active ester in the figure), thereby generating a conjugate of the compound of the present invention and the antibody [an antibody that position-selectively has a structural unit comprising an affinity substance for the antibody and a cleavage moiety: ALB-R'-T (II)]. Figure 1-2 This is a schematic diagram (part 2) illustrating the concept of position-selective modification of an antibody using the compound of the present invention. Cleavage of the cleavable moiety in the linker (L) generates an antibody that is position-selectively modified with a bioorthogonal functional group.

[0602] Figure 1-3 This is a schematic diagram (part 3) illustrating the concept of position-selective modification of antibodies using the compounds of the present invention. Through the reaction of bioorthogonal functional groups with functional substances (e.g., drugs), antibodies modified specifically with functional substances are generated.

[0603] Figure 2 : This is a diagram showing the correlation between the various inventions of the present invention (notations for salts are omitted). In reaction (1), a compound having an affinity substance for an antibody, a cleavage portion, and a reactive group is reacted with an antibody to generate an antibody having an affinity substance for the antibody and a cleavage portion. In reaction (2), an antibody having an affinity substance for the antibody and a cleavage portion is reacted with a functional substance to generate a complex having an affinity substance for the antibody, a cleavage portion, a functional substance, and the antibody. In reaction (3), the cleavage portion of the complex having an affinity substance for the antibody, a cleavage portion, a functional substance, and the antibody is cleaved to generate an antibody having a functional substance (in this case, a portion containing the affinity substance is generated as a by-product). In reaction (4), the cleavage portion of the antibody having an affinity substance for the antibody and a cleavage portion is cleaved to generate an antibody having a bioorthogonal functional group (in this case, a portion containing the affinity substance is generated as a by-product). In reaction (5), the antibody having a bioorthogonal functional group reacts with a functional substance to generate an antibody having a functional substance. Reactions (2) and (5) can be performed in the same manner. In addition, reactions (3) and (5) can also be performed in the same manner.

[0604] Figure 3A diagram showing the consensus amino acid sequence (SEQ ID NO: 1) of the Fc region of the heavy chain of trastuzumab and the IgG1 Fc region;

[0605] Figure 4 This figure shows (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 2), (2) the amino acid sequence of the IgG1 Fc region after cleavage of the sugar chain with PNGase (SEQ ID NO: 3), and (3) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 4);

[0606] Figure 5 Graph showing secretory expression of various modified proteins A in Corynebacterium glutamicum (C. glutamicum). Lane 1: Molecular weight marker, Lane 2: Protein band speculated to be CspB6Tev-QZ34C, Lane 3: Protein band speculated to be QZ34C-60, Lane 4: Protein band speculated to be QET-Z34C1, Lane 5: Protein band speculated to be QET-Z34C2.

[0607] Figure 6 This figure shows the results of HIC (Hydrophobic Interaction Chromatography)-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 3). The vertical axis (AU) represents absorbance (the same applies to the following figures);

[0608] Figure 7 This is a graph showing the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 4);

[0609] Figure 8 This is a graph showing the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 5);

[0610] Figure 9 This figure shows the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 6);

[0611] Figure 10This figure shows the MS spectrum (measured: m / z 952.23196, calculated: 952.22900, tetravalent) of a peptide fragment of trastuzumab consisting of 33 amino acid residues, the peptide THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 26), containing a modification site on a lysine residue (thiol-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) after trypsin digestion (Example 7);

[0612] Figure 11-1 This is a diagram showing the CID spectrum of the product ion of m / z 764.68 (theoretical value: 764.40) corresponding to the divalent y12, indicating modification of the lysine residues at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 7);

[0613] Figure 11-2 : This is a graph showing the results of searching for peptide fragments containing modifications to lysine residues (sulfhydryl groups introduced by carbamidomethylation using iodoacetamide (+145.019 Da)) using Proteome Discoverer (Thermo Fisher Scientific) for trypsin digests of trastuzumab (Example 7). The horizontal axis represents the identified lysine residues, and the vertical axis represents the peptide spectrum match map (PSM, Peptide Spectrum Match). The residue numbering of lysine residues is represented by the numbering in the sequence (i.e., the N-terminal amino acid is the first position, the same below) for the heavy chain VH domain and the light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains.

[0614] Figure 12 This figure shows the MS spectrum (measured: m / z 952.23227, calculated: 952.22900, tetravalent) of a peptide fragment of trastuzumab consisting of 33 amino acid residues, the peptide THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 26), containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation using iodoacetamide) after trypsin digestion (Example 8);

[0615] Figure 13-1 This is a diagram showing the CID spectrum of the product ion of m / z 838.23 (theoretical value: 837.94) corresponding to the divalent y13, indicating modification of the lysine residues at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 8);

[0616] Figure 13-2 This figure shows the results of searching for peptide fragments containing modifications to lysine residues (sulfhydryl groups introduced by carbamidomethylation using iodoacetamide (+145.019 Da)) using Proteome Discoverer (Thermo Fisher Scientific) for trypsin digests of trastuzumab (Example 8). The horizontal axis represents the identified lysine residues, and the vertical axis represents the peptide spectrum match map (PSM). The residue numbering of lysine residues is represented by the sequence numbering for the heavy chain VH domain and light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains.

[0617] Figure 14 This figure shows the MS spectrum (measured: m / z 952.23032, calculated: 952.22900, tetravalent) of a peptide fragment of trastuzumab consisting of 33 amino acid residues, the peptide THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 26), containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) after trypsin digestion (Example 9);

[0618] Figure 15-1 This is a diagram showing the CID spectrum of the product ion of m / z 838.29 (theoretical value: 837.94) corresponding to the divalent y13, which shows modification of the lysine residues at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 9);

[0619] Figure 15-2 This figure shows the results of searching for peptide fragments containing modifications to lysine residues (sulfhydryl groups introduced by carbamidomethylation using iodoacetamide (+145.019 Da)) using Proteome Discoverer (Thermo Fisher Scientific) for trypsin digests of trastuzumab (Example 9). The horizontal axis represents the identified lysine residues, and the vertical axis represents the peptide spectrum match map (PSM). The residue numbering of lysine residues is represented by the sequence numbering for the heavy chain VH domain and light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains.

[0620] Figure 16 Graph showing the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm);

[0621] Figure 17 This is a graph showing the MS spectrum (m / z 791.74872, trivalent) of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 47) containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binder peptide in Reference Example 1, digested with trypsin;

[0622] Figure 18 A diagram showing the CID spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 47) containing a modified site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binding peptide in Reference Example 1, digested with trypsin;

[0623] Figure 19 A graph showing the MS spectrum (m / z 886.93408, tetravalent) of a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (SEQ ID NO: 48) containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binding peptide in Reference Example 1, digested with trypsin;

[0624] Figure 20 A diagram showing the CID spectrum of a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (SEQ ID NO: 48) containing a modified site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binding peptide in Reference Example 1, digested with trypsin;

[0625] Figure 21 This is a graph showing the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Reference Example 3);

[0626] Figure 22 This is a graph showing the MS spectrum (m / z 769.04688, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 50) containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binding peptide according to Reference Example 3, digested with trypsin;

[0627] Figure 23 A diagram showing the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 50) containing a modified site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) of trastuzumab modified with a binding peptide in Reference Example 3, digested with trypsin;

[0628] Figure 24 This is a diagram showing SDS-PAGE of the secretory expression of each modified protein A (QET-Z34C3 and QET-Z34C4) in Corynebacterium glutamicum (C. glutamicum);

[0629] Figure 25 This is a diagram showing SDS-PAGE of the secretory expression of each modified Fc-III (CspBss-QET-LV and CspBss-QET-II) in Corynebacterium glutamicum (C. glutamicum);

[0630] Figure 26 This graph shows the MS spectrum of a peptide fragment of trastuzumab (FNWYVDGVEVHNAKTKPR (SEQ ID NO: 50), an 18-amino acid residue peptide containing a modified site on a lysine residue (thiol group-introduced form (+145.019 Da) after carbamidomethylation with iodoacetamide) digested with trypsin (measured: m / z 769.04353, calculated: 769.04482, trivalent).

[0631] Figure 27-1 This is a diagram showing the CID spectrum of the product ion of m / z 1022.73 (theoretical value: 1022.51) corresponding to the divalent y16, indicating modification of the lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering (Example 12);

[0632] Figure 27-2 This figure shows the results of searching for peptide fragments containing modified lysine residues (thiol groups introduced by carbamidomethylation using iodoacetamide (+145.019 Da)) using BioPharma Finder for a tryptic digest of trastuzumab (Example 12). The horizontal axis represents the identified lysine residues, and the vertical axis represents the intensity. The residue numbers of lysine residues are represented by the sequence numbering (i.e., the N-terminal amino acid is the first position, the same below) for the heavy chain VH domain and light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains.

[0633] Figure 28 This is a diagram showing the confirmation of specific modified forms of trastuzumab based on ESI-TOFMS analysis (Example 13);

[0634] Figure 29 This is a graph showing the results of HIC-UPLC analysis of specific modification of trastuzumab (detection wavelength: UV 280 nm) (Example 13);

[0635] Figure 30 This figure shows the MS spectrum (measured: m / z 952.23425, calculated: 952.22942, tetravalent) of a peptide fragment of trastuzumab consisting of 33 amino acid residues, the peptide THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 26), containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) after trypsin digestion (Example 14);

[0636] Figure 31 This figure shows the MS spectrum (measured: m / z 952.23425, calculated: 952.22942, tetravalent) of a peptide fragment of trastuzumab consisting of 33 amino acid residues, the peptide THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 26), containing a modification site on a lysine residue (thiol group-introduced form (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) after trypsin digestion (Example 14);

[0637] Figure 32-1 This is a diagram showing the CID spectrum of the product ion of m / z 1166.99 (theoretical value: 1166.61) corresponding to the divalent y20, indicating modification of the lysine residues at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 14);

[0638] Figure 32-2 This figure shows the results of a BioPharma Finder search for peptide fragments containing modified lysine residues (thiol-introduced products (+145.019 Da)) of a tryptic digest of trastuzumab (Example 14). The horizontal axis represents the identified lysine residues, and the vertical axis represents intensity. Lysine residues are numbered using the sequence numbering (i.e., the N-terminal amino acid is considered the first position, the same applies below) for the heavy chain VH domain and light chain, and EU numbering for the heavy chain CH1, CH2, and CH3 domains. DETAILED DESCRIPTION

[0639] 1. Compound or salt thereof comprising an affinity substance for an antibody, a cleavable moiety, and a reactive group 1-1. Overview

[0640] The present invention provides a compound represented by formula (I) comprising an affinity substance for an antibody, a cleavable portion and a reactive group, or a salt thereof;

[0641] ALBR (I)

[0642] Where,

[0643] A is an affinity substance for antibodies,

[0644] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[0645] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0646] R is a reactive group toward the antibody.

[0647] In formula (I) and other formulae shown in the present invention, a hyphen (-) indicates that the two units present on either side thereof are covalently bonded (covalently bonded). Therefore, in formula (I), A is covalently bonded to L, L is covalently bonded to A and B, B is covalently bonded to L and R, and R is covalently bonded to B.

[0648] 1-2. Affinity substances for antibodies (A)

[0649] In formula (I), A is an affinity substance for an antibody. An affinity substance refers to a substance that has the ability to bind to an antibody by non-covalent binding.

[0650] The affinity substance used in the present invention is targeted at antibodies. Antibodies can be proteins (such as glycoproteins) modified with biomolecules (such as sugars), or proteins that are not modified with biomolecules. As antibodies, any antibodies against any components such as components derived from organisms, components derived from viruses, and components found in the environment can be used, but antibodies against components derived from organisms or components derived from viruses are preferred. As components derived from organisms, for example, components (such as proteins) derived from animals such as mammals, birds (such as chickens), insects, microorganisms, plants, fungi, and fish can be listed. Components derived from organisms are preferably components derived from mammals. As mammals, for example, primates (such as humans, monkeys, chimpanzees), rodents (such as mice, rats, guinea pigs, hamsters, rabbits), pet animals (such as dogs, cats), livestock (such as cattle, pigs, goats), and working animals (such as horses, sheep). Components derived from organisms are more preferably components (such as proteins) derived from primates or rodents. From the perspective of the clinical application of the present invention, components (such as proteins) derived from humans are further preferred. Examples of virus-derived components include components (eg, proteins) derived from influenza virus (eg, avian influenza virus, swine influenza virus), HIV, Ebola virus, and bacteriophage virus.

[0651] In addition, the antibody is a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. As a monoclonal antibody, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies with the addition of prescribed sugar chains (for example, antibodies modified in the manner of sugar chains with N-type sugar chains binding consensus sequences, etc.), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab') 2 antibodies, VHH antibodies, Fc regional proteins, Fc fusion proteins can be listed. The antibody can also be a divalent antibody (such as IgG, IgD, IgE) or a tetravalent or higher antibody (such as IgA antibody, IgM antibody).

[0652] The antibody as the target of the affinity substance can also be composed of any amino acid residues, preferably composed of the 20 natural L-α-amino acid residues that generally constitute proteins. Examples of such amino acid residues include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and L-glycine (G) (hereinafter, L is omitted). The antibody can be composed of, for example, more than 100, preferably more than 120, more preferably more than 150, further more preferably more than 180, and particularly preferably more than 200 amino acid residues. The antibody can also be, for example, less than 1000, preferably less than 900, more preferably less than 800, further more preferably less than 700, and particularly preferably less than 600. More specifically, the antibody can be composed of, for example, 100 to 1000, preferably 120 to 900, more preferably 150 to 800, further more preferably 180 to 700, and particularly preferably 200 to 600 amino acid residues. When the antibody is an antibody (such as the above-mentioned monoclonal antibody), the above number of amino acid residues can be equivalent to the amino acid residues of the heavy chain of the antibody.

[0653] The antibody as the target of affinity substance can also be a protein containing a side chain or terminal (N-terminal and / or C-terminal) that can react with a reactive group as described later, preferably a specific amino acid residue of a side chain at one position or multiple positions (preferably multiple positions). As such a specific amino acid residue, for example, 14 kinds of amino acid residues as described later can be enumerated, preferably an amino acid residue selected from lysine residues, tyrosine residues, tryptophan residues and cysteine ​​residues. Such a specific amino acid residue is more preferably an amino acid residue selected from lysine residues, tyrosine residues and tryptophan residues. If it is considered that the compound of the present invention can positionally modify antibodies, it is preferably an antibody containing such a specific amino acid residue at multiple positions. As multiple positions, as long as it is more than 2 positions, it is not particularly limited, for example, it can be more than 3 positions, preferably more than 5 positions, more preferably more than 10 positions, further more preferably more than 20 positions, and particularly preferably more than 30 positions. The plurality of positions may also be, for example, positions of 200 or less, preferably positions of 180 or less, more preferably positions of 150 or less, further preferably positions of 120 or less, and particularly preferably positions of 100 or less. More specifically, the plurality of positions may be, for example, 3 to 200 positions, preferably 5 to 180 positions, more preferably 10 to 150 positions, further preferably 20 to 120 positions, and particularly preferably 30 to 100 positions. Even for antibodies comprising specific amino acid residues at multiple positions, the compounds of the present invention may be selectively modified at specific amino acid residue positions present at a specific position. For example, the number of lysine residues in human IgG1 also varies depending on the amino acid composition in the variable region and is generally considered to be about 70 to 90. In the present invention, such positional selective modification of lysine residues present at specific positions of human IgG1 has been successfully achieved.

[0654] More specifically, in the present invention, from the perspective of modifying the amino acid residues at specific positions in the antibody while maintaining the function of the antibody (i.e., maintaining the native folding without denaturing the antibody), it is preferred to selectively modify the amino acid residues exposed on the surface of the antibody. For example, in human IgG1 and other human IgG, exposed lysine residues and exposed tyrosine residues are present at the following positions (based on EU numbering, refer to http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). Among them, in this application, for ease of description, EU numbering is used for the heavy chain CH1, CH2, and CH3 domains, and the numbering in the sequence (i.e., the N-terminal amino acid is the first position) is used for the heavy chain VH domain and the light chain;

[0655] (1) Exposed lysine residues

[0656] CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322, 338) CH3 domain (positions 360, 414, 439)

[0657] (2) Exposed tyrosine residues

[0658] CH2 domain (positions 278, 296, and 300)

[0659] CH3 domain (position 436)

[0660] Therefore, when human IgG1 or other human IgG is modified at a lysine residue or a tyrosine residue, the modification is preferably at the above position.

[0661] Preferably, when a lysine residue or tyrosine residue is modified in human IgG1 or other human IgG, the lysine residue or tyrosine residue that is modified in the positions (1) and (2) above may be a position that is highly exposed on the surface or below;

[0662] (1') exposed lysine residues

[0663] CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322)

[0664] CH3 domain (positions 360, 414, and 439)

[0665] (2') exposed tyrosine residues

[0666] CH2 domain (positions 278, 296, and 300)

[0667] CH3 domain (position 436)

[0668] Therefore, when human IgG1 or other human IgG has a lysine residue or a tyrosine residue modified, the modification is preferably at the above position.

[0669] More preferably, when the lysine residue of human IgG such as human IgG1 is modified, the lysine residue at the position (1) above may be a specified position (e.g., position 246, position 248, position 288, position 290, position 317) in the CH2 domain that can be effectively modified in the present invention.

[0670] In a specific embodiment, the antibody as the target of the affinity substance, when containing specific amino acid residues at multiple positions as described above, may be a target region formed by 1 to 50 consecutive amino acid residues containing more than one specific amino acid residue, and a non-target region other than the target region containing more than 5 specific amino acid residues. The target region may be formed by preferably 1 to 30, more preferably 1 to 20, further preferably 1 to 10, 1 to 5, or 1 to 3 (i.e., 1, 2 or 3) amino acid residues. It is particularly preferred that the target region be a region formed by specific amino acid residues present at a specific position. Such a specific position also varies according to the type of target protein and affinity substance, for example, it may be a specific position in a specific region (e.g., CH1, CH2, CH3) in the constant region of an antibody, preferably a position in CH2 of an antibody. More specifically, the target region may be the following residues based on EU numbering in human IgG Fc:

[0671] (1) Lys248 residue (hereinafter also referred to as "Lys248" in this specification, corresponding to residue 18 in the CH2 region of human IgG (SEQ ID NO: 1)) or Lys246 residue (hereinafter also referred to as "Lys246" in this specification, corresponding to residue 16 in the CH2 region of human IgG (SEQ ID NO: 1));

[0672] (2) Lys288 residue (hereinafter also referred to as “Lys288” in this specification, corresponding to residue 58 of the human IgG CH2 region (SEQ ID NO: 1)) or Lys290 residue (hereinafter also referred to as “Lys290” in this specification, corresponding to residue 60 of the human IgG CH2 region (SEQ ID NO: 1));

[0673] (3) Lys317 residue (hereinafter also referred to as "Lys317" in this specification, corresponding to residue 87 in the human IgG CH2 region (SEQ ID NO: 1)).

[0674] According to the present invention, specific amino acid residues in the target region can be modified with high positional selectivity. Such positional selectivity can be, for example, 30% or greater, preferably 40% or greater, more preferably 50% or greater, even more preferably 60% or greater, particularly preferably 70% or greater, 80% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100% or greater.

[0675] The target region may also be a region extending from the N-terminal side and the C-terminal side to a position separated by a (where a is an arbitrary integer of 1 to 10) amino acid residues from a specific amino acid residue present at a specific position, with the specific amino acid residue at the specific position as the center, and containing no amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position. a is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, further preferably 1 or 2, and particularly preferably 1.

[0676] In a preferred embodiment, the antibody is a monoclonal antibody. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Monoclonal antibodies are full-length antibodies or antibody fragments (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibodies), preferably full-length antibodies.

[0677] Antibodies are antibodies directed against any antigen. For example, such antigens may be components found in organisms or viruses as described above. Examples of such antigens include proteins (including oligopeptides and polypeptides. Proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds.

[0678] The antibody may preferably be an antibody having a protein as an antigen. Examples of the protein include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.

[0679] More specifically, the protein serving as an antigen for the antibody may be a disease-targeting protein. Examples of disease-targeting proteins include the following.

[0680] (1) Cancer field

[0681] PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endothelial growth factor), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (insulin-like growth factor factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, Glypican-3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, tissue factor Factor), IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor-associated macrophages,tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor (tissue factor) factor), EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis blood group B antigen, A34, Lysyl-Oxidase, DLK-1, TROP-2, Integrin α9, TAG-72 (CA72-4), CD70.

[0682] (2) Autoimmune diseases-inflammatory diseases

[0683] IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin) Lymphopoietin), LAMP (integrin α4β7), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 ligand, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, integrin α9, LIFHT.

[0684] (3) Brain and nerve diseases

[0685] CGRP, CD20, β-amyloid protein, β-amyloid fibrils, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus.

[0686] (4) Infectious diseases

[0687] Clostridium Difficile toxin B, Cytomegalovirus, RS virus, LPS, S.Aureus Alpha-toxin, M2e protein, Psl, PcrV, S.Aureus toxin, Influenza A, Alginate, Staphylococcus aureus, PD-L1, Influenza B, Acinetobacter, F protein, Env, CD3, Pathogenic Escherichia coli, Klebsiella pneumoniae, Pneumococcus.

[0688] (5) Hereditary-rare diseases

[0689] Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, Huntington's disease.

[0690] (6) Eye diseases

[0691] Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (endoglin), IGF-1R, and β-amyloid protein.

[0692] (7) Orthopedics and Plastic Surgery

[0693] Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15.

[0694] (8) Blood diseases

[0695] vWF, factor IXa, factor X, IFNγ, C5, BMP-6, ferroportin, TFPI.

[0696] (9) Other diseases

[0697] BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP.

[0698] In a more preferred embodiment, the affinity substance for the antibody is an affinity substance for a monoclonal antibody. The isotype of the monoclonal antibody is the same as that described above for the antibody, preferably IgG (e.g., IgG1, IgG2, IgG3, IgG4). Preferably, the monoclonal antibody is a full-length monoclonal antibody.

[0699] In a further more preferred embodiment, the substance having affinity for an antibody is a chimeric antibody, a humanized antibody, or a human antibody (eg, IgG such as IgG1, IgG2, IgG3, or IgG4) which is a full-length monoclonal antibody.

[0700] In a particularly preferred embodiment, the affinity substance for an antibody is an affinity substance for the following antibody, wherein the antibody comprises any one of the Fc region proteins selected from the following (A) to (C) and has antigen-binding ability:

[0701] (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1;

[0702] (B) an Fc region protein comprising an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted into the amino acid sequence of SEQ ID NO: 1; or

[0703] (C) An Fc region protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1.

[0704] The amino acid sequence of SEQ ID NO: 1 is an Fc region protein. Such Fc region proteins are known to have secretory ability. Therefore, the Fc region proteins of (A) to (C) above may have secretory ability. In addition, antibodies comprising such Fc region proteins may have antigen binding ability. The amino acid residue at position 18 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain as described below, further preferably leucine, isoleucine or alanine, particularly preferably leucine or alanine. The amino acid residue at position 19 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue or an acidic amino acid residue, more preferably an amino acid residue having a non-polar side chain or an acidic amino acid residue, further preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, further preferably glycine or alanine. The amino acid residue at position 140 in SEQ ID NO: 1 is any amino acid residue, preferably an acidic amino acid residue, more preferably glutamic acid or aspartic acid. The amino acid residue at position 142 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, further preferably methionine, leucine, or isoleucine, particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO: 1 is any amino acid residue, preferably a neutral amino acid residue, more preferably an amino acid residue having an uncharged polar side chain or an amino acid residue having a non-polar side chain as described below, further preferably threonine, alanine, or glycine, particularly preferably threonine or alanine.

[0705] In a preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence formed by amino acid residues at positions 220 to 449 in the amino acid sequence of SEQ ID NO: 2.

[0706] In another preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence formed by amino acid residues at positions 7 to 236 in the amino acid sequence of SEQ ID NO: 3.

[0707] In a specific embodiment, an antibody comprising an "Fc region protein comprising the above-described amino acid sequence" may be an antibody comprising an "Fc region protein comprising the above-described amino acid sequence" and an "antibody constant region." Such an antibody constant region may be a chimeric antibody, a humanized antibody, or a human antibody (e.g., an IgG such as IgG1, IgG2, IgG3, or IgG4).

[0708] In the Fc region protein (B), one or several amino acid residues can be changed by one, two, three or four mutations selected from the deletion, substitution, addition and insertion of amino acid residues. The mutation of the amino acid residue can be introduced into one region in the amino acid sequence, or it can be introduced into multiple different regions. The term "one or several" means the number that does not significantly destroy the activity of the protein. The number shown in the term "one or several" is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (for example, 1, 2, 3, 4 or 5).

[0709] In the Fc region protein (C), the % identity with the amino acid sequence of SEQ ID NO: 1 can be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more. In the present invention, the % identity of the peptide or polypeptide (protein) can be calculated by the algorithm blastp. More specifically, the calculation of the % identity of the polypeptide can be performed using the default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11 Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment) in the algorithm blastp provided by the National Center for Biotechnology Information (NCBI). In addition, the calculation of the % identity of the polynucleotide (gene) can be performed by the algorithm blastn. More specifically, the calculation of the % identity of the polynucleotide can be performed using the default scoring parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear) in the blastn algorithm provided by NCBI.

[0710] The term "secretion" in the secretory capacity has the same meaning as the secretion of secretory proteins (so-called solubility). Therefore, "having secretory capacity" means that the protein functions as an antibody in the same manner as a normal antibody.

[0711] The above-mentioned antibody comprising the Fc region protein, as long as the target characteristic (such as secretory ability, antigen binding ability) is maintained, can be introduced with variation at a specific position. The position of the amino acid residue that can be introduced into the variation that can maintain the target characteristic is self-evident to those skilled in the art. Specifically, for those skilled in the art, 1) compare the amino acid sequences of various proteins with the same characteristics, 2) clarify the relatively conservative (preserved) region and the relatively non-conserved region, then 3) the relatively conservative region and the relatively non-conserved region can be respectively predicted to play an important role for the function and the region that cannot play an important role for the function, so the correlation between structure and function can be identified. Therefore, those skilled in the art can specify the position of the amino acid residue that can be introduced into the amino acid sequence of the above-mentioned antibody comprising the Fc region protein.

[0712] In the case where an amino acid residue mutates by substitution, the substitution of an amino acid residue can be a conservative substitution. In the case used in this specification, the term "conservative substitution" refers to the substitution of a prescribed amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such a family includes amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids having side chains containing hydroxyl groups (e.g., alcoholic, phenolic) (e.g., serine, threonine, tyrosine), and amino acids having sulfur-containing side chains (e.g., cysteine, methionine). Preferred conservative substitutions of amino acids may be substitutions between aspartic acid and glutamic acid, substitutions between arginine, lysine and histidine, substitutions between tryptophan and phenylalanine, substitutions between phenylalanine and valine, substitutions between leucine, isoleucine and alanine, and substitutions between glycine and alanine.

[0713] Examples of antibodies comprising an Fc region selected from any one of (A) to (C) above include chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, and altertoxaximab), humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab), and alemtuzumab). uzumab), Omalizumab, Efalizumab, Bevacizumab, Natalizumab (IgG4), Tocilizumab, Eculizumab (IgG2), Mogamulizumab, Pertuzumab, Obinutuzumab, Vedolizumab, Pembrolizumab (IgG4), Mepolizumab lizumab), Elotuzumab, Daratumumab, Ixekizumab (IgG4), Reslizumab (IgG4), Atezolizumab), human antibodies (e.g., Adalimumab (IgG1), Panitumumab, Golimumab, Ustekinumab, Canakinumab, Ofatumumab, Denosumab), mab) (IgG2), Ipirimumab, Belimumab, Raxibacumab, Ramucirumab, Nivolumab (IgG4), Secukinumab, Evolocumab (IgG2), Alirocumab, Necitumumab, Brodalumab (IgG2), Olaratumab) (IgG1 is indicated if IgG isotype is not mentioned).

[0714] In the present invention, the affinity substance for antibodies is a polypeptide containing a glutamine residue (Q) at its N-terminus. By using a polypeptide containing a glutamine residue (Q) at its N-terminus as an affinity substance for antibodies, the amino group (-NH2) in the N-terminal glutamine residue (Q) can be protected by reacting the amino group (-NH2) with the amide group present in its side chain (pyroglutamylation). Therefore, a linker that mediates the binding of the affinity substance for antibodies can be specifically added to a specific amino acid residue (lysine residue having an amino group) in the affinity substance.

[0715] Examples of polypeptides containing a glutamine residue (Q) at the N-terminus include polypeptides obtained by adding a "glutamic acid residue" or a "peptide containing a glutamic acid residue at the N-terminus" to the N-terminus of an affinity polypeptide for an antibody. Various polypeptides have been reported as affinity polypeptides for antibodies (e.g., International Publication No. 2016 / 186206, International Publication No. 2013 / 027796, International Publication No. 2008 / 054030, International Publication No. 2001 / 045746, Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, Vol. 6, 564-569; Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, Vol. 49, 443-454; Ruvo M et al., ChemBioChem, 2005, Vol. 6, 1242-1253; Krook M et al., Journal of Immunological Methods, 1998, Vol. 221, 151-157; Jacobs et al., J. Biochem. Biophys. Methods, 2001, Vol. 49, 443-454). JM et al., Bio.Techniques, 2003, VOL.34, 132-141; Carbonell RG et al., Journal of Chromatography A, 2009, VOL.1216, 910-918; Lund LN et al., Journal of Chromatography A, 2012, VOL.1225, 158-167; Warren L. Delano et al., Science, 2000, VOL.287, 1279-1283; Biochemical Engineering Journal, 2013, VOL.79, 33-40).

[0716] As the polypeptide comprising glutamine residues (Q) at the N-terminus, it is better to use the polypeptide prepared by the secretion production method (International Publication No. 2013 / 062029) of the polypeptide using coryneform bacteria as a host. The method can add the N-terminal three residues Gln-Glu-Thr (QET) of the Csp mature protein at the N-terminus of the target polypeptide, and can simply and in large quantities prepare the polypeptide comprising glutamine residues (Q) at the N-terminus, so it is suitable for the preparation of the affinity material for antibody. As the coryneform bacteria that can be used in the method, for example: coryneform bacteria (Corynebacterium) genus bacteria (for example, Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium stationis (Corynebacterium stationis)) and Brevibacterium (Brevibacterium) genus bacteria.

[0717] The number of amino acid residues in a polypeptide containing a glutamine residue (Q) at the N-terminus is not particularly limited, but from the perspective of secretory production efficiency in a secretory production method of a polypeptide using a coryneform bacterium as a host, it can be 16 or more and 123 or less. Preferably, the number of amino acid residues can be 20 or more, 25 or more, 30 or more, 35 or more, 37 or more, or 40 or more. Alternatively, the number of amino acid residues can be 73 or less, 68 or less, 64 or less, 61 or less, 58 or less, or 53 or less.

[0718] In a specific embodiment, the polypeptide comprising a glutamine residue (Q) at the N-terminus may be a polypeptide selected from the following (1) to (4);

[0719] [Chemical Formula 16]

[0720]

[0721] Q is the N-terminal glutamine residue, E is a glutamic acid residue, T is a threonine residue, the solid line is a bond, the spacer portion is a portion formed by one or more and 50 or less amino acid residues, and the affinity substance for the antibody is a portion formed by 15 or more and 70 or less amino acid residues.

[0722] In the polypeptides of (1) to (4) above, the number of amino acid residues in the spacer region is preferably 1 or more and 40 or less, more preferably 1 or more and 30 or less, further preferably 1 or more and 20 or less, and particularly preferably 1 or more and 10 or less. Any amino acid residue may be used as the amino acid residue, but the 20 standard L-α-amino acids constituting natural proteins are preferred.

[0723] In the polypeptides of (1) to (4) above, the number of amino acid residues in the affinity polypeptide portion for antibodies is 15 or more and 70 or less, preferably 15 or more, 17 or more, 20 or more, 25 or more, 30 or more, 34 or more, 35 or more, or 40 or more, and preferably 65 or less, 61 or less, 60 or less, 55 or less, or 50 or less. In the polypeptides of (1) to (4) above, any affinity polypeptide for antibodies (e.g., the polypeptides reported above) can be used as the affinity polypeptide portion for antibodies, preferably selected from protein A, protein G, protein L, protein Z, fragments thereof having affinity for antibodies, and mutants thereof having affinity for antibodies.

[0724] Protein A is a non-glycosylated protein (Staphylococcus aureus Protein A: SpA) present in the cell wall of Staphylococcus aureus. It is known that the multiple domains of Protein A (domains A, B, C, D, and E) each have affinity for antibodies (e.g., Moks T et al., Eur J Biochem. 1986 May 2; 156(3): 637-43). Therefore, polypeptides corresponding to such domains can be used as fragments of Protein A. More specifically, the polypeptide corresponding to such domains has the following amino acid sequence (58 to 61 amino acid residues);

[0725] (1) Domain A: ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 27)

[0726] (2) Domain B: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 28)

[0727] (3) Domain C: ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 29)

[0728] (4) Domain D: ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK (SEQ ID NO: 30)

[0729] (5) Domain E: AANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK (SEQ ID NO: 31).

[0730] Protein G is a protein present in the cell wall of group G streptococci. It is known that multiple domains of protein G (domains C2 and C3) each have affinity for antibodies (e.g., Sjobring UJ et al., Biol Chem. 1991 Jan 5; 266(1): 399-405). Therefore, polypeptides corresponding to such domains can be used as fragments of protein G. More specifically, the polypeptide corresponding to such domains has the following amino acid sequence (60 amino acid residues);

[0731] (1) Domain C2: TPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 32)

[0732] (2) Domain C3: TPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE (SEQ ID NO: 33).

[0733] Protein L is a protein derived from Peptostreptococcus magnus. It is known that multiple domains of protein L (domains C, C1 to C4, and B1 to B5) have affinity for antibodies (e.g., Graille M et al., Structure. 2001 Aug;9(8):679-87). Therefore, polypeptides corresponding to such domains can be used as fragments of protein L. More specifically, the polypeptide corresponding to such domains has the following amino acid sequence (61 amino acid residues);

[0734] (1) Domain C: EVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGNHMNIKFA (SEQ ID NO: 34)

[0735] (2) Domain C1: EVTIKANLIFADGSTQNAEFKGTFAKAVSDAYAYADALKKDNGEYTVDVADKGLTLNIKFA (SEQ ID NO: 35)

[0736] (3) Domain C2: EVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFA (SEQ ID NO:36)

[0737] (4) Domain C3: EVTIKVNLIFADGKIQTAEFKGTFEEATAKAYAYANLLAKENGEYTADLEDGGNTINIKFA (SEQ ID NO:37)

[0738] (5) Domain C4: EVTIKVNLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGYTINIKFA (SEQ ID NO:38)

[0739] (6) Domain B1: EVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFA (SEQ ID NO:39)

[0740] (7) Domain B2: EVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFA (SEQ ID NO:40)

[0741] (8) Domain B3: EVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFA (SEQ ID NO:41)

[0742] (9) Domain B4: EVTIKANLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFA (SEQ ID NO:42)

[0743] (10) Domain B5: QVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTINIRFA (SEQ ID NO:43).

[0744] Protein Z is an artificial IgG binding polypeptide designed based on the above-mentioned protein A (e.g., Nilsson B et al., Protein Eng. 1987 Feb-Mar; 1(2): 107-13.). Protein Z has the following amino acid sequence (58 amino acid residues). Z34C is an artificial IgG binding polypeptide formed by a full length of 34 amino acid residues, which is designed based on the above-mentioned protein A in the same manner as protein Z (e.g., Starovasnik MA et al., Proc Natl Acad Sci USA. 1997 Sep 16; 94(19): 10080-5). Regarding Z34C, multiple amino acid residue substitutions were introduced into protein Z in order to improve the affinity for antibodies, but because the full length of amino acids is shorter than that of protein Z, it can also be considered to be a fragment of protein Z. Therefore, a shortened polypeptide of protein Z (e.g., an N-terminal and / or C-terminal shortened polypeptide formed by at least 34 amino acid residues) and Z34C can be used as a fragment of protein Z;

[0745] (1) Protein Z: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 44)

[0746] (2) Z34C: FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO: 45).

[0747] A fragment of protein A, protein G, protein L, or protein Z with affinity for antibodies is a shortened polypeptide obtained by deleting the amino acid residues at the N-terminal side and / or the C-terminal side of protein A, G, L, or Z. It is known that in protein A, G, L, or Z, affinity for antibodies does not require all of the amino acid residues constituting them, and even if the amino acid residues at the N-terminal side and / or the C-terminal side are deleted, affinity for antibodies (e.g., those having the above-mentioned domains) is still present. Therefore, one skilled in the art can, by making a shortened polypeptide obtained by deleting the amino acid residues at the N-terminal side and / or the C-terminal side of protein A, G, L, or Z, and then evaluating whether the shortened polypeptide thus prepared has affinity for antibodies, thereby suitably preparing a fragment of protein A, G, L, or Z with affinity for antibodies (a fragment of protein A, G, L, or Z with affinity for antibodies).

[0748] As a mutant of protein A, protein G, protein L or protein Z or their fragments with affinity for antibodies, for example, homologous polypeptides having an amino acid sequence identity of more than 60% and having affinity for antibodies with protein A, protein G, protein L or protein Z or their fragments can be enumerated. It is known that even homologous polypeptides with such an amino acid sequence identity also have affinity for antibodies (for example, Starovasnik MA et al., Proc Natl Acad Sci US A. 1997 Sep 16; 94 (19): 10080-5). The amino acid sequence identity can be preferably more than 70%, more preferably more than 80%, further more preferably more than 90%, particularly preferably more than 95% (for example, more than 96%, more than 97%, more than 98% or more than 99%). Amino acid sequence identity can be calculated in the same manner as the identity % of the above-mentioned polypeptide.

[0749] In a specific embodiment, the affinity polypeptide portion for an antibody is a fragment of protein A or a mutant thereof having affinity for the antibody (a fragment of protein A or a mutant thereof having affinity for the antibody). Preferably, the fragment of protein A or a mutant thereof having affinity for the antibody may comprise: an amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNAXIXSIRDDC (SEQ ID NO: 5) (where each of the two Xs is independently an amino acid residue selected from an arginine residue, a glutamic acid residue, and a glutamine residue), in which any one amino acid residue is substituted with a lysine residue, and an amino acid sequence having 90% or greater identity with the amino acid sequence of SEQ ID NO: 5 (in an amino acid sequence having 90% or greater identity with the amino acid sequence of SEQ ID NO: 5, the substitution at the lysine residue is maintained). In other words, such a polypeptide portion may be: (i) a polypeptide portion in which any amino acid residue in the amino acid sequence of SEQ ID NO:5 is substituted with a lysine residue; or (ii) a polypeptide portion in which any amino acid residue in the amino acid sequence of SEQ ID NO:5 is substituted with a lysine residue and 1 to 3 amino acid residues other than the lysine residue are substituted with other amino acid residues, or other amino acid residues are inserted or added. Preferably, the amino acid sequence of SEQ ID NO:5 may be the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:6).

[0750] In a preferred embodiment, the antibody-binding polypeptide is a mutant of a protein A fragment having affinity for antibodies, with a glutamine residue (Q) or QET added to the N-terminus. Examples of such mutants include the following polypeptides:

[0751] (1)QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7);

[0752] (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQID NO: 8);

[0753] (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9);

[0754] (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10);

[0755] (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22);

[0756] (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23);

[0757] (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51);

[0758] (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); and

[0759] (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53).

[0760] In another specific embodiment, the affinity polypeptide for an antibody can be a modified peptide (modified Fc-III) of the peptide Fc-III (WO2001 / 045746) composed of the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 54), which is reported to have the ability to bind to human IgG. Such a modified Fc-III is, for example, the following polypeptide:

[0761] (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and

[0762] (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

[0763] At least two cysteine ​​residues separated by a disulfide bond in each amino acid sequence of the affinity polypeptide may form a cyclic peptide. Alternatively, the sulfide groups of the two cysteines in the peptide may be linked by a carbonyl-containing linker as shown below.

[0764] [Chemical Formula 17]

[0765]

[0766] The dashed portion of the carbonyl-containing linker shown above represents the portion bound to the thioether group. This linker is more stable than a typical disulfide bond in reactions such as reduction. Such peptides can be prepared, for example, by the method described in International Publication No. 2016 / 186206.

[0767] A novel peptide having such a specific structure can be used for position-selective modification of Lys248 or Lys246 based on Eu numbering in human IgG Fc, or other amino acid residues other than Lys248 or Lys246 (Examples). The amino acids constituting the above peptides are either L- or D-initiators, but are preferably L-initiators (all amino acid residues constituting the peptides in the Examples are L-initiators).

[0768] The above-mentioned peptide may be modified with a cross-linking agent at a specific amino acid residue. Examples of such a specific amino acid residue include a lysine residue, an aspartic acid residue, and a glutamic acid residue, with a lysine residue being preferred. Examples of the cross-linking agent include DSG (disuccinimidyl glutarate, disuccinimidyl glutarate) and DSS (disuccinimidyl suberate, disuccinimidyl suberate), which preferably contain two or more succinimide groups; DMA (dimethyl adipimidate·2HCl, dimethyl diimidate dihydrochloride), DMP (dimethyl pimelimidate·2HCl, dimethyl pimelimidate dihydrochloride), and DMS (dimethylsuberimidate·2HCl, dimethyl suberimidate dihydrochloride), which preferably contain two or more imidic acid moieties; and DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl, dimethyl 3,3'-dithiobispropionimidate dihydrochloride) and DSP (dithiobis(succinimidyl)propionimidate dihydrochloride). propionate), dithiobis(succinimidylpropionate)) and the like (for example, International Publication No. 2016 / 186206).

[0769] Regarding the above-mentioned peptides, the amino and carboxyl groups at the terminals may be protected. Examples of the protecting group for the N-terminal amino group include alkylcarbonyl (acyl) groups (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkoxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl groups, and arylalkyl (aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). An acetyl group is preferably used as the protecting group for the N-terminal amino group. Examples of the protecting group for the C-terminal carboxyl group include groups that can form esters or amides. Examples of the groups that can form esters or amides include alkoxy groups (e.g., methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy groups), aryloxy groups (e.g., phenoxy, naphthyloxy groups), aralkyloxy groups (e.g., benzyloxy), and amino groups. An amino group is preferably used as the protecting group for the C-terminal carboxyl group.

[0770] 1-3. Connector (L)

[0771] In formula (I), L is a cleavable linker which is a divalent group containing a cleavable moiety.

[0772] The cleavable linker represented by L is a divalent group containing a cleavable portion. The cleavable portion is a site (part) that can be cleaved by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of an amide bond). Therefore, the cleavable portion can be a site (bond other than an amide bond) that is cleaved by a specific cleavage treatment under mild conditions. Examples of such specific treatments include: (a) treatment with one or more substances selected from acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with a physicochemical stimulus selected from light, or (c) placement using a cleavable linker containing a self-degradable cleavable portion. Such cleavable linkers and their cleavage conditions are common technical knowledge in this field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society. 132, 179-28 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)). Examples of such cleavable moieties include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tert-alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residues), silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, residues containing an aromatic ring having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, chain residues containing unsaturated bonds, and glycosyl residues.

[0773] The aromatic ring group with an electron-withdrawing group is preferably a group having an aromatic ring group selected from an aryl group, an aralkyl group, an aromatic heterocyclic group, an alkyl group with an aromatic heterocyclic group, more preferably an aralkyl group, an alkyl group with an aromatic heterocyclic group. The electron-withdrawing group is preferably bound to the 2-position of the ring. More preferably, the residue containing the aromatic ring with an electron-withdrawing group is, for example, an aralkyl group (such as a benzyl group) having an electron-withdrawing group at the 2-position. As the electron-withdrawing group, for example, a halogen atom, an alkyl group substituted with a halogen atom (such as a trifluoromethyl group), a boronic acid residue, a methanesulfonyl group, a toluenesulfonyl group, a trifluoromethanesulfonate or an ester (trifluoromethanesulfonyl group), a nitro group, a cyano group, a phenyl group, a keto group (such as an acyl group) can be listed.

[0774] The definitions, examples and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, and aralkyl used as linkers and suffixes in connection with the names of the residues of the cleavable portion are the same as those described below.

[0775] Examples of the ester residue include conventional ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tert-alkyloxycarbonyl groups such as tert-butoxycarbonyl), aryl esters (e.g., phenacyl ester, 2-(diphenylphosphino)benzoate), glycosyl ester residues, orthoester residues], ester residues containing sulfur and oxygen atoms (e.g., thioester residues such as α-thiophenyl ester residues and alkylthio ester residues), ester residues containing phosphorus and oxygen atoms (e.g., phosphodiester residues, phosphotriester residues), and active ester residues (e.g., N-hydroxysuccinimide residues).

[0776] Examples of the sulfone-containing residue include a sulfone residue and a quinolylbenzenesulfonate residue.

[0777] The silane residue is preferably a silane residue having a group selected from an alkyl group, an aryl group, an aralkyl group, and an alkoxy group. Examples of such silane residues include dialkyldialkoxysilane residues (e.g., dimethyldialkoxysilane, diethyldialkoxysilane), and diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

[0778] As an alkoxyalkyl (i.e., alkyloxyalkyl) residue, a group obtained by combining an alkoxy group and an alkyl group described later (the definitions, examples, and preferred examples of alkoxy and alkyl groups are the same as those described later), for example, a methoxymethyl residue, an ethoxymethyl residue, a methoxyethyl residue, and an ethoxyethyl residue can be mentioned, but it is not limited to these examples.

[0779] A chain residue containing an unsaturated bond is a residue containing an unsaturated bond portion formed only by carbon atoms (e.g., a vinyl group (vinyl / ethenyl) as the smallest unit having a double bond between carbon atoms, or an acetylenyl group (acetylenyl / ethynyl) as the smallest unit having a triple bond between carbon atoms), or a residue containing an unsaturated bond portion formed by a carbon atom and a heteroatom (e.g., a nitrogen atom, a sulfur atom, or an oxygen atom) (e.g., an aldehyde group or a cyano group). Examples of chain residues containing an unsaturated bond include a vinyl ether residue, a cyanoethyl residue, an ethylene residue, and a malondialdehyde residue.

[0780] Examples of acidic substances (also referred to as electrophilic reagents) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinepropanesulfonic acid, sodium dihydrogen phosphate, citric acid, dodecylsulfuric acid, N-lauroylsarcosine, and trifluoroacetic acid. Examples of sites that can be cleaved by acidic substances include alkoxyarylalkyl residues, tert-alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, β-thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphino)benzoate residues.

[0781] Examples of alkaline substances (also referred to as nucleophiles) include inorganic alkaline substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, and ammonium acetate, and organic alkaline substances such as hydroxylamine, triethylamine, and N,N'-diisopropylamine. Examples of sites that can be cleaved by alkaline substances include silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyl disuccinate residues, α-thiophenyl ester residues, unsaturated vinyl sulfide residues, malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

[0782] Examples of the reducing agent include cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Examples of the site cleavable by the reducing agent include a disulfide residue, an alkoxyalkyl residue, and an azo residue.

[0783] Examples of the oxidizing agent include sodium periodate and oxidized glutathione, and examples of the site cleavable by the oxidizing agent include a vicinal diol residue and a selenium residue.

[0784] Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspases, proteases, matrix metalloproteinases, lipases, endoglycosidases, and peptide N-glycosidase F (PNGase F). Examples of sites (parts) that can be cleaved by the enzyme include ester residues, phosphodiester residues, and glycosyl residues.

[0785] Examples of the photocleavable site include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinolbenzenesulfonate residue, a coumarin residue, a phosphotriester residue, a bisarylhydrazone residue, and a Bimandithiopropionic acid residue.

[0786] Examples of the self-degradable cleavable moiety include active ester residues (eg, N-hydroxysuccinimide residues).

[0787] More specifically, the cleavable moiety may correspond to any chemical structure selected from the following group;

[0788] [Chemical Formula 18]

[0789]

[0790] Here, the wavy line orthogonal to the bond indicates the cleavage site,

[0791] Multiple R 2a , multiple R 2b and multiple R 2c are the same or different and are selected from a hydrogen atom or a substituent described below,

[0792] J is -CH2-, -O- or -S-,

[0793] r is an arbitrary integer from 1 to 4,

[0794] ○ (white circle) represents the bond to A (or La described later), and ● (black circle) represents the bond to B (or Lb described later). When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A (or La described later), and ○ may represent the bond to B (or Lb described later).

[0795] J is -CH2-, -O- or -S-. J is preferably -CH2- or -O-, more preferably -CH2-.

[0796] r is any integer of 1 to 4, preferably any integer of 1 to 3, more preferably 1 or 2.

[0797] In one embodiment, the cleavable linker can be:

[0798] (i) a cleavable linker, which is a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the side of the reactive group by cleavage; or

[0799] (ii) A cleavable linker, which is a divalent group comprising a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side by cleavage.

[0800] Examples of the cleavable moiety (i) include a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue.

[0801] More specifically, the cleavable moiety of (i) may correspond to, for example, any chemical structure selected from the following group;

[0802] [Chemical Formula 19]

[0803]

[0804] Here, the wavy line orthogonal to the bond indicates the cleavage site,

[0805] Multiple R 2a are the same or different and are selected from a hydrogen atom or a substituent described below,

[0806] ○ (white circle) represents the bond to A (or La described later), and ● (black circle) represents the bond to B (or Lb described later). When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A (or La described later), and ○ may represent the bond to B (or Lb described later).

[0807] Examples of the cleavable moiety (ii) include an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue.

[0808] More specifically, the cleavable moiety of (ii) may correspond to, for example, any chemical structure selected from the following group;

[0809] [Chemical Formula 20]

[0810]

[0811] Here, the wavy line orthogonal to the bond indicates the cleavage site,

[0812] Multiple R2b , multiple R 2c , J, r are selected from hydrogen atoms or substituents described below,

[0813] ○ (white circle) represents the bond to A (or La described later), and ● (black circle) represents the bond to B (or Lb described later). When the chemical structure is asymmetric around the cleavage site, ● may represent the bond to A (or La described later), and ○ may represent the bond to B (or Lb described later).

[0814] In a specific embodiment, the cleavable linker (L) can be represented by any one of the following formulae (L1) to (L3); La-C-Lb (L1)

[0815] La-C (L2)

[0816] C-Lb (L3)

[0817] Where,

[0818] La and Lb are divalent groups,

[0819] C is the cutting part.

[0820] Examples of the divalent group include a divalent hydrocarbon group which may have a substituent, a divalent heterocyclic group which may have a substituent, -C(=O)-, -NR a -(R a represents a hydrogen atom or a substituent), -O-, -S-, -C(=S)-, and a combination of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) thereof.

[0821] The divalent hydrocarbon group is a linear, branched or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of the divalent hydrocarbon group include an alkylene group, an alkenylene group, an alkynylene group and an arylene group.

[0822] The alkylene group is preferably an alkylene group having 1 to 12 carbon atoms, more preferably an alkylene group having 1 to 6 carbon atoms, and particularly preferably an alkylene group having 1 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. The alkylene group may be linear, branched, or cyclic, but is preferably a linear alkylene group. Examples of such alkylene groups include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0823] The alkenylene group is preferably an alkenylene group having 2 to 12 carbon atoms, more preferably an alkenylene group having 2 to 6 carbon atoms, and particularly preferably an alkenylene group having 2 to 4 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in substituents. The alkenylene group may be straight-chain, branched, or cyclic, but is preferably a straight-chain alkenylene group. Examples of such alkenylene groups include ethenylene, propenylene, butenylene, pentenylene, and hexenylene.

[0824] The alkynylene group is preferably an alkynylene group having 2 to 12 carbon atoms, more preferably an alkynylene group having 2 to 6 carbon atoms, and particularly preferably an alkynylene group having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. The alkynylene group may be straight-chain, branched, or cyclic, but is preferably a straight-chain alkynylene group. Examples of such alkynylene groups include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0825] The arylene group is preferably an arylene group having 6 to 24 carbon atoms, more preferably an arylene group having 6 to 18 carbon atoms, further preferably an arylene group having 6 to 14 carbon atoms, and still further preferably an arylene group having 6 to 10 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0826] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it is preferred to include one or more selected from an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include one or more selected from an oxygen atom, a sulfur atom and a nitrogen atom.

[0827] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 1 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 1 to 15 carbon atoms, further preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and still further preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in the substituent. More specifically, examples of the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazoldiyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0828] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, further preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and still further preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in the substituent. More specifically, examples of the divalent non-aromatic heterocyclic group include a pyrroledione diyl group, a pyrrolidinedione diyl group, an oxiranediyl group, an aziridinediyl group, an azetidinediyl group, an oxetanediyl group, a thietanediyl group, a pyrrolidinediyl group, a dihydrofurandiyl group, a tetrahydrofurandiyl group, a dioxolanediyl group, a tetrahydrothiophenediyl group, a pyrrolidinediyl group, an imidazolidinediyl group, an oxazolidinediyl group, a piperidinediyl group, a dihydropyrandiyl group, a tetrahydropyrandiyl group, a tetrahydrothiopyrandiyl group, a morpholinediyl group, a thiomorpholinediyl group, a pyrazinediyl group, a dihydrooxazinediyl group, a tetrahydrooxazinediyl group, a dihydropyrimidinediyl group, and a tetrahydropyrimidinediyl group.

[0829] The divalent groups represented by La and Lb may have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents. a and R b As such substituents, for example, the following groups can be cited:

[0830] (i) halogen atoms,

[0831] (ii) a monovalent hydrocarbon group,

[0832] (iii) an aralkyl group,

[0833] (iv) a monovalent heterocyclic group,

[0834] (v)R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or a monovalent hydrocarbon group, or (vi) NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e The same or different, represents a hydrogen atom or a monovalent hydrocarbon group,

[0835] (vii) nitro, sulfate, sulfonate, cyano and carboxyl groups.

[0836] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0837] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0838] A monovalent chain hydrocarbon group refers to a hydrocarbon group consisting solely of a chain structure, with the main chain not containing a cyclic structure. The chain structure may be straight-chain or branched. Examples of monovalent chain hydrocarbon groups include alkyl groups, alkenyl groups, and alkynyl groups. Alkyl groups, alkenyl groups, and alkynyl groups may be straight-chain or branched.

[0839] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and still more preferably an alkyl group having 1 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the alkyl group having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0840] The alkenyl group is preferably an alkenyl group having 2 to 12 carbon atoms, more preferably an alkenyl group having 2 to 6 carbon atoms, and still more preferably an alkenyl group having 2 to 4 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in substituents. Examples of the alkenyl group having 2 to 12 carbon atoms include ethenyl, propenyl, and n-butenyl.

[0841] The alkynyl group is preferably an alkynyl group having 2 to 12 carbon atoms, more preferably an alkynyl group having 2 to 6 carbon atoms, and even more preferably an alkynyl group having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the alkynyl group having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0842] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0843] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group whose ring structure contains only alicyclic hydrocarbons and no aromatic rings. The alicyclic hydrocarbons may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; a portion thereof may contain a chain structure. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl groups, cycloalkenyl groups, and cycloalkynyl groups. These groups may be monocyclic or polycyclic.

[0844] The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 carbon atoms, more preferably a cycloalkyl group having 3 to 6 carbon atoms, and still more preferably a cycloalkyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of the substituent. Examples of the cycloalkyl group having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0845] The cycloalkenyl group is preferably a cycloalkenyl group having 3 to 12 carbon atoms, more preferably a cycloalkenyl group having 3 to 6 carbon atoms, and still more preferably a cycloalkenyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the cycloalkenyl group having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0846] The cycloalkynyl group is preferably a cycloalkynyl group having 3 to 12 carbon atoms, more preferably a cycloalkynyl group having 3 to 6 carbon atoms, and still more preferably a cycloalkynyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the cycloalkynyl group having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0847] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0848] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. It need not be composed solely of aromatic rings; a portion thereof may contain a chain structure or an alicyclic hydrocarbon, and the aromatic ring may be monocyclic or polycyclic. The monovalent aromatic hydrocarbon group is preferably an aryl group having 6 to 12 carbon atoms, more preferably an aryl group having 6 to 10 carbon atoms, and even more preferably an aryl group having 6 carbon atoms. The aforementioned number of carbon atoms does not include the number of carbon atoms in the substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0849] The monovalent aromatic hydrocarbon group is preferably a phenyl group.

[0850] Among them, the monovalent hydrocarbon group is preferably an alkyl group, a cycloalkyl group or an aryl group, more preferably an alkyl group.

[0851] Aralkyl refers to an arylalkyl group. The definitions, examples, and preferred embodiments of the aryl and alkyl groups in the arylalkyl group are as described above. Aralkyl groups preferably have 3 to 15 carbon atoms. Examples of such aralkyl groups include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0852] A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from a heterocyclic ring of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic group preferably include one or more selected from oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably include one or more selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0853] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and still more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in the substituent. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinoneyl, carbazolyl, fluorenyl, quinolyl, isoquinolyl, quinazolinyl, and phthalazinyl.

[0854] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and still more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the monovalent non-aromatic heterocyclic group include oxirane, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0855] Among them, the monovalent heterocyclic group is preferably a 5-membered or 6-membered heterocyclic group.

[0856] Preferably, the substituents may be the following groups:

[0857] (i') a halogen atom,

[0858] (ii') an alkyl group having 1 to 12 carbon atoms, a phenyl group or a naphthyl group,

[0859] (iii') an aralkyl group having 3 to 15 carbon atoms,

[0860] (iv') a 5-membered or 6-membered heterocyclic ring,

[0861] (v')R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms, or

[0862] (vi')NR d R e -、NR d R e -C(=O)-, NRd R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or an alkyl group having 1 to 12 carbon atoms,

[0863] (vii') The same groups as exemplified in the above (vii).

[0864] More preferably, the substituent may be a group:

[0865] (i") halogen atoms,

[0866] (ii") an alkyl group having 1 to 12 carbon atoms,

[0867] (iii”)R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms, or

[0868] (iv”)NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or an alkyl group having 1 to 12 carbon atoms,

[0869] (v") The same groups as exemplified in the above (vii).

[0870] More preferably, the substituent may be the following group:

[0871] (i'') halogen atoms,

[0872] (ii'') an alkyl group having 1 to 6 carbon atoms,

[0873] (iii”')R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R crepresents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, or

[0874] (iv”')NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[0875] (v''") are the same groups as exemplified in the above-mentioned (vii).

[0876] Particularly preferably, the substituent may be the following group:

[0877] (i””) halogen atoms,

[0878] (ii””) an alkyl group having 1 to 4 carbon atoms,

[0879] (iii””)R c -O-、R c -C(=O)-、R c -OC(=O)- or R c -C(=O)-O-,R c represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, or

[0880] (iv””)NR d R e -、NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -, R d and R e are the same or different and represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

[0881] (v") The same groups as exemplified in the above (vii).

[0882] In certain embodiments, La and Lb may be represented by (La') and (Lb'), respectively, as follows:

[0883] [Chemical Formula 21]

[0884]

[0885] Where,

[0886] p and p' are the same or different and are any integer from 0 to 10,

[0887] q and q' are the same or different and are any integer from 0 to 10,

[0888] X and X' are the same or different and are carbon atoms, nitrogen atoms or single bonds (where X is a nitrogen atom, R 1b does not exist; when X' is a nitrogen atom, R 1b' Does not exist. When X is a single bond, R 1a and R 1b Does not exist; when X' is a single bond, R 1a' and R 1b' does not exist),

[0889] R 1a 、R 1b 、R 1a' and R 1b' are the same or different and are selected from a hydrogen atom or the substituents mentioned above.

[0890] p and p' are the same or different and are any integer of 0 to 10, preferably an integer of 0 to 8, more preferably an integer of 0 to 6, further preferably an integer of 0 to 4, particularly preferably 0, 1 or 2. It is preferred that p and p' are the same.

[0891] q and q' are the same or different and are any integer of 0 to 10, preferably an integer of 0 to 8, more preferably an integer of 0 to 6, further preferably an integer of 0 to 4, particularly preferably 0, 1 or 2. q and q' are preferably the same.

[0892] X and X' are the same or different and are carbon atoms, nitrogen atoms or single bonds, preferably carbon atoms or single bonds. X and X' are preferably the same.

[0893] R 1a 、R 1b 、R 1a' and R 1b' are the same or different and are selected from hydrogen atoms or substituents described below. The definitions, examples and preferred examples of the substituents are as described above. Preferably, R 1a 、R 1b 、R 1a' and R 1b' A hydrogen atom.

[0894] 1-4. (a) A divalent group containing a bioorthogonal functional group, or (b) A divalent group not containing a bioorthogonal functional group (B)

[0895] In formula (I), B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group.

[0896] Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0897] When the target of the affinity substance is an antibody, the bioorthogonal functional group is a bioorthogonal functional group for proteins. A bioorthogonal functional group for proteins is a group that does not react with the side chains of the 20 naturally occurring amino acid residues that make up a protein, but instead reacts with a predetermined functional group. The 20 naturally occurring amino acids that make up a protein are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., their side chains do not contain heteroatoms selected from sulfur, nitrogen, and oxygen atoms), are inert to typical reactions. Therefore, bioorthogonal functional groups for proteins are those that do not react with the side chains of these amino acids having side chains inert to typical reactions, and also do not react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0898] Examples of bioorthogonal functional groups that do not react with proteins include azide residues, aldehyde residues, thiol residues, olefin residues (in other words, any residue having a vinyl moiety as the smallest unit having a double bond between carbon atoms, the same below), alkyne residues (in other words, any residue having an ethynyl moiety as the smallest unit having a triple bond between carbon atoms, the same below), halogen residues, tetrazine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester groups, α-halogenated carbonyl residues (for example, a carbonyl residue having a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom at the α-position, the same below), isonitrile residues, sterone residues, and selenium residues. Proteins include proteins that may contain free thiol groups (e.g., proteins other than antibodies) and proteins that do not contain free thiol groups (e.g., antibodies). In proteins that do not contain free sulfhydryl groups, sulfhydryl groups can function as bioorthogonal functional groups. Therefore, since the target of affinity substances is antibodies that do not contain free sulfhydryl groups, the bioorthogonal functional groups include sulfhydryl groups. The divalent group may contain one or more (e.g., two, three, or four) bioorthogonal functional groups, but preferably, one bioorthogonal functional group is contained in the divalent group.

[0899] In one embodiment, the divalent group containing a bioorthogonal functional group can be: a divalent group containing a bioorthogonal functional group selected from an azide residue, an aldehyde group, a thiol residue, an alkyne residue, an olefin residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halogenated carbonyl residue, an isonitrile residue, a sterone residue, and a selenium residue in the main chain.

[0900] In another embodiment, the divalent group containing a bioorthogonal functional group can be: a divalent group containing a bioorthogonal functional group selected from an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an olefin residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, an α-halogenated carbonyl residue, an isonitrile residue, a sterone residue, and a selenium residue in the side chain.

[0901] More specifically, the bioorthogonal functional group may correspond to any chemical structure selected from the following group:

[0902] [Chemical Formula 22]

[0903]

[0904] Where,

[0905] R 1f , single or multiple R 1g , and single or multiple R 1h are the same or different and are atoms or groups selected from (i) to (vii), or electron-withdrawing groups,

[0906] ·Indicates a bonding key.

[0907] Examples of the electron-withdrawing group include the above-mentioned groups, and a halogen atom, a boronic acid residue, a methanesulfonyl group, a toluenesulfonyl group, a trifluoromethanesulfonate or an ester is preferred.

[0908] In one embodiment, B may be (a) a divalent group containing a bioorthogonal functional group. The number of bioorthogonal functional groups contained in the divalent group is one or more, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and further preferably 1. When the number of bioorthogonal functional groups contained in the divalent group is multiple, the multiple bioorthogonal functional groups may be of the same type or different types, but from the perspective of adopting a simple structure, they are preferably of the same type.

[0909] In a specific embodiment, B can be (a1) a divalent group containing a bioorthogonal functional group in the main chain. The divalent group containing a bioorthogonal functional group in the main chain is: a group having a bioorthogonal functional group selected from azide residue, aldehyde group, thiol residue, alkyne residue, olefin residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halogenated carbonyl residue, isonitrile residue, sterone residue, and selenium residue as a divalent group, or a group having the above-mentioned divalent group connected to one end or both ends of such a divalent bioorthogonal functional group. The definition, examples, and preferred examples of the divalent group to be connected are the same as those of the divalent group described above.

[0910] In another specific embodiment, B can be (a2) a divalent group containing a bioorthogonal functional group in the side chain. The divalent group containing a bioorthogonal functional group in the side chain is a divalent group substituted with a bioorthogonal functional group selected from azide residues, aldehyde residues, thiol residues, alkyne residues, olefin residues, halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, α-halogenated carbonyl residues, isonitrile residues, sterone residues, and selenium residues, or a group containing the same. The definition, examples, and preferred embodiments of the divalent group to be substituted are the same as those of the divalent group described above.

[0911] In another embodiment, B may be (b) a divalent group that does not contain a bioorthogonal functional group. Such a divalent group may be: a substituted alkylene group, a substituted cycloalkylene group, a substituted aryl group, a substituted divalent heterocyclic group, -NR a -(R a represents a hydrogen atom or a substituent), -O-, and a group formed by a combination of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of these groups. Substituents and R in the case of substitution a The substituents are substituents other than bioorthogonal functional groups. Examples of such substituents include alkyl groups, cycloalkyl groups, aralkyl groups, monovalent heterocyclic groups, hydroxyl groups, amino groups, alkyloxy groups (alkoxy groups), cycloalkyloxy groups, and aralkyloxy groups. The number of such substituents is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

[0912] Regarding substituents other than the bioorthogonal functional group, the definitions, examples, and preferred examples of the alkyl group, cycloalkyl group, aralkyl group, and monovalent heterocyclic group are as described above.

[0913] Regarding substituents other than bioorthogonal functional groups, the definitions, examples, and preferred examples of the alkyl group in an alkyloxy group (alkoxy), the cycloalkyl group in a cycloalkoxy group, and the aralkyl group in an aralkyloxy group are as described above. More specifically, examples of the alkoxy group include methoxy, ethoxy, propoxy (e.g., n-propoxy, isopropoxy), butoxy (e.g., n-butoxy, isobutoxy, sec-butoxy, tert-butoxy), pentoxy (e.g., n-pentoxy), and hexoxy (e.g., n-hexoxy). Examples of cycloalkyloxy groups include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Examples of aralkyloxy groups include benzyloxy, phenethoxy, naphthylmethoxy, and naphthylethoxy.

[0914] The divalent group not containing a bioorthogonal functional group can also be a group that is highly inert to the reaction. Therefore, such a divalent group can be a group consisting only of carbon atoms and hydrogen atoms. Such a divalent group is an alkylene group, a cycloalkylene group or an aryl group and a combination of two or more (for example, 2 or 3) of these groups. In the case where the divalent group not containing a bioorthogonal functional group is a group that is highly inert to the reaction, such a divalent group may have a substituent selected from an alkylene group, a cycloalkylene group and an aryl group as a substituent that is highly inert to the reaction. The number of substituents that are highly inert to the reaction is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

[0915] In a specific embodiment, B can be represented by the following formula (B-1):

[0916] [Chemical Formula 23]

[0917]

[0918] Where,

[0919] Y is -NH-, -O-, -CH2- or the following formula (B-2),

[0920] [Chemical Formula 24]

[0921]

[0922] (In formula (B-2),

[0923] V and V' are the same or different and are -NH-, -O-, -CH2- or a single bond,

[0924] V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0925] s is an arbitrary integer from 0 to 10,

[0926] In formula (B-2), ○ and ● are in the same orientation as ○ and ● in formula (B-1),

[0927] Z is an oxygen atom, a sulfur atom or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH2-),

[0928] In formula (B-1), ○ (white circle) represents a bond bonded to the moiety on the L side, and ● (black circle) represents a bond bonded to the moiety on the R side.

[0929] Y is -NH-, -O-, -CH2-, or a group represented by the above formula (B-2). From the perspective of structural simplification, Y may be -NH-, -O-, or -CH2-. Alternatively, from the perspective of designing a structure based on carbon atoms, Y may be -CH2- or a group represented by the above formula (B-2).

[0930] Z is an oxygen atom, a sulfur atom or a hydrogen atom, preferably an oxygen atom or a sulfur atom.

[0931] V and V' are -NH-, -O-, -CH2- or a single bond, preferably -CH2- or a single bond.

[0932] V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group. Such a divalent group is the same as the above-mentioned group.

[0933] Preferably, the divalent group in V1 is a substitutable divalent hydrocarbon group or a substitutable divalent heterocyclic group. The definition, examples and preferred examples of the divalent hydrocarbon group are the same as those described above. In the case of V1, it is preferably an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group, or an arylene group. For example, when not substituted with a portion containing a bioorthogonal functional group, it is preferably an alkenylene group, an alkynylene group, a cycloalkenylene group, or a cycloalkynylene group. On the other hand, when substituted with a portion containing a bioorthogonal functional group, it is preferably an alkylene group, a cycloalkylene group, or an arylene group. Examples and preferred examples of these groups are as described above. The definition, examples and preferred examples of the divalent heterocyclic group are the same as those described above. In the case of V1, it is preferably a 5-membered or 6-membered heterocyclic group. Examples and preferred examples of the 5-membered or 6-membered heterocyclic group are the same as those described above. The definition, examples and preferred examples of the substituent are as described above. V1 may have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and further preferably 1 (a) bioorthogonal functional groups. When the number of bioorthogonal functional groups contained in the divalent group is multiple, the multiple bioorthogonal functional groups may be of the same type or of different types. From the viewpoints of adopting a simple structure and improving reactivity, it is preferably of the same type. From the viewpoint of ensuring differentiated reactions, it is preferably of different types. V1 may also have 1 to 5, preferably 1 to 3, and more preferably 1 or 2 (b) substituents.

[0934] s is any integer of 0 to 10, preferably an integer of 0 to 8, more preferably an integer of 0 to 6, further preferably an integer of 0 to 4, particularly preferably 0, 1 or 2.

[0935] In addition, in a specific embodiment, V1 may be a divalent group having a group represented by the following formula (B-3) as a side chain;

[0936] [Chemical Formula 25]

[0937]

[0938] Where,

[0939] G and G' are the same or different and are -NH-, -O-, -CH2-, a single bond or a group represented by the following formula (B-4),

[0940] [Chemical Formula 26]

[0941]

[0942] In formula (B-4),

[0943] W and W' are the same or different and are -NH-, -O-, -CH2- or a single bond,

[0944] W1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[0945] t is any integer from 0 to 10,

[0946] In formula (B-4), ○ (white circle) indicates a bond (bond) in the direction of the bond (·) in formula (B-3), and ● (black circle) indicates a bond in the direction of the b side;

[0947] H is -CH2-, -C=O-, -C=S-, -NH- or a single bond;

[0948] I is a divalent hydrocarbon group, a divalent heterocycle or a single bond;

[0949] b is any of the following groups,

[0950] [Chemical Formula 27]

[0951]

[0952] Where,

[0953] R 1f , single or multiple R 1g , and single or multiple R 1h are the same or different and are atoms or groups selected from (i) to (vii), or electron-withdrawing groups,

[0954] · represents a bonding bond. Such a divalent group is a divalent hydrocarbon group or a divalent heterocyclic group, preferably a substitutable divalent hydrocarbon group, more preferably an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, a cycloalkynylene group or an arylene group, further preferably an alkylene group, a cycloalkylene group or an arylene group, and particularly preferably an alkylene group. Examples and preferred examples of these groups are as described above. These groups may be substituted with substituents other than the above-mentioned side chains. The number of such substituents is 1 to 5, preferably 1 to 3, more preferably 1 or 2. Examples and preferred examples of substituents are as described above.

[0955] G and G' are the same or different and are -NH-, -O-, -CH2-, a single bond, or a group represented by the above formula (B-4). From the perspective of structural simplification, G and G' may be -NH-, -O-, -CH2-, or a single bond. Alternatively, from the perspective of designing a structure based on carbon atoms, G and G' may be -CH2-, a single bond, or a group represented by the above formula (B-4).

[0956] H is -CH2-, -C=O-, -C=S-, -NH-, or a single bond. Preferably, H is -CH2- or a single bond.

[0957] I is a divalent hydrocarbon group, a divalent heterocycle, or a single bond. The divalent hydrocarbon group and the divalent heterocycle may be substituted or unsubstituted. The definitions, examples, and preferred embodiments of the divalent hydrocarbon group, the divalent heterocycle, and the substituent are the same as those described in V1 above.

[0958] W and W' are the same or different and are -NH-, -O-, -CH2- or a single bond, preferably -CH2- or a single bond.

[0959] W1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group. Such divalent groups are the same as those described above.

[0960] t is any integer of 0 to 10, preferably an integer of 0 to 8, more preferably an integer of 0 to 6, further preferably an integer of 0 to 4, particularly preferably 0, 1 or 2.

[0961] 1-5. Reactive Group (R)

[0962] In formula (I), R is a reactive group for antibodies. Such reactive groups are common technical knowledge in this technical field.

[0963] The reactive group is a group of the same or different type as the bioorthogonal functional group.

[0964] For example, when B is (a) a divalent group containing a bioorthogonal functional group, the reactive group may be a group of a different species from the bioorthogonal functional group. This is because if the reactive group is the same species as the bioorthogonal functional group, the reactive specificity for the reactive group in the antibody cannot be ensured. Furthermore, bioorthogonal functional groups are groups that do not react with the side chains of the 20 naturally occurring amino acid residues that constitute antibodies.

[0965] More specifically, among the 20 natural amino acids that constitute proteins, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups, are inert to typical reactions. Therefore, the reactive group for proteins is a group that can react with any one or more (e.g., two, three, or four) of the side chains of the 14 amino acids: asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Depending on conditions such as the amino acid composition of the protein, the compound represented by formula (I) may contain one or more (e.g., two, three, or four) reactive groups, and preferably, the compound represented by formula (I) may contain one reactive group.

[0966] Preferably, the reactive group is a group that can react with the side chain of any one of the 14 amino acids constituting proteins as described above.

[0967] More preferably, the reactive group may be a reactive group specific for the side chain of any one of lysine, tyrosine, tryptophan, and cysteine.

[0968] Even more preferably, the reactive group may be a reactive group specific to the side chain of any one of lysine, tyrosine, and tryptophan.

[0969] Furthermore, when the protein is human IgG1 or other human IgG, the reactive group is preferably a reactive group specific for the side chain of lysine or tyrosine.

[0970] The reactive group specific for the side chain of the lysine residue is a group that can specifically react with the amino group (NH2) present in the side chain of the lysine residue, and examples thereof include active ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, and diazoterephthalic acid residues.

[0971] Examples of the linking portion generated by the reaction between the "reactive group specific for the side chain of the lysine residue" and the "amino group (NH2) present in the side chain of the lysine residue" include an amide residue, a urea residue, a pyridine residue, a carbamate residue, and a sulfonamide residue.

[0972] More specifically, the reactive group specific for the side chain of a lysine residue may correspond to any chemical structure selected from the following group;

[0973] [Chemical Formula 28]

[0974]

[0975] Here,

[0976] R 5a and R 5c is a hydrogen atom or the above-mentioned substituent,

[0977] R 5b is an electron-withdrawing group,

[0978] j is an arbitrary integer from 1 to 5,

[0979] k is an arbitrary integer from 1 to 4.

[0980] R5a and R 5c is a hydrogen atom or the above-mentioned substituent. The definitions, examples and preferred examples of the substituent are the same as those described above.

[0981] R 5b Examples of such electron-withdrawing groups include those mentioned above, and preferably include a halogen atom, a boronic acid residue, a methanesulfonyl group, a toluenesulfonyl group, a trifluoromethanesulfonate or an ester.

[0982] j is any integer of 1-5, preferably an integer of 1-3, more preferably 1 or 2.

[0983] k is an arbitrary integer of 1-4, preferably an integer of 1-3, more preferably 1 or 2.

[0984] The linking portion generated by the reaction between the "above-mentioned chemical structure as a reactive group specific for the side chain of the lysine residue" and the "amino group (NH2) present in the side chain of lysine" may correspond to any chemical structure selected from the following group;

[0985] [Chemical Formula 29]

[0986]

[0987] Here, ● (black circle) represents a bond bonded to the T-side portion, ○ (white circle) represents a bond bonded to the B-side portion, and straight lines perpendicular to the bonds represent bonds generated by the reaction.

[0988] The reactive group specific for the side chain of the tyrosine residue is a group that can react specifically with an atom adjacent to the phenolic hydroxyl group (OH) present in the side chain of the tyrosine residue, and examples thereof include a diazo residue, an azodicarboxylate residue, and a 2,3-dihydro-1H-pyrazin-6-one residue.

[0989] More specifically, the reactive group specific for the side chain of a tyrosine residue may correspond to any chemical structure selected from the following group;

[0990] [Chemical formula 30]

[0991]

[0992] Here, R 4a represents a hydrogen atom or the above-mentioned substituent, and ○ represents a bond with B.

[0993] R 4a is a hydrogen atom or the above-mentioned substituent. The definitions, examples and preferred examples of the substituent are the same as those described above.

[0994] The linking portion generated by the reaction between the “above-mentioned chemical structure as a reactive group specific for the side chain of the tyrosine residue” and the “atom adjacent to the phenolic hydroxyl group (OH) present in the side chain of the tyrosine residue” may correspond to any chemical structure selected from the following group;

[0995] [Chemical Formula 31]

[0996]

[0997] Here, R 4a represents a hydrogen atom or the above-mentioned substituent, ● represents a bond to T, and ○ represents a bond to B.

[0998] R 4a is a hydrogen atom or the above-mentioned substituent. The definitions, examples and preferred examples of the substituent are the same as those described above.

[0999] The reactive group specific for the side chain of the tryptophan residue is a group that can react specifically with the ring-forming atom at the 3-position of the indole group present in the side chain of the tryptophan residue, and examples thereof include a 9-azabicyclo[3.3.1]nonan-3-one-N-oxyl radical residue.

[1000] More specifically, the reactive group specific for the side chain of a tryptophan residue may correspond to any chemical structure selected from the following group;

[1001] [Chemical Formula 32]

[1002]

[1003] Here, ○ represents a bond with B.

[1004] The linking portion generated by the reaction between the "above-mentioned chemical structure as a reactive group specific for the side chain of the tryptophan residue" and the "ring-forming atom at position 3 of the indole group present in the side chain of the tryptophan residue" may correspond to any chemical structure selected from the following group;

[1005] [Chemical Formula 33]

[1006]

[1007] Here, ● represents a bond to T, and ○ represents a bond to B.

[1008] Particularly preferably, the reactive group may be a reactive group specific for the side chain of lysine.

[1009] 1-6. Partial structure "LB" 1-6-1. Length of the main chain in the partial structure "LB" connecting A and R In formula (I), the length of the main chain (straight chain portion in LB) connecting A (affinity substance) and R (reactive group) can be appropriately designed according to various factors such as the type of antibody and affinity substance, and the relationship between the target site of the affinity substance in the antibody and the position and number of specific amino acid residues in the target area (e.g., a specific position) to which R is to react and bind. With respect to the compound represented by formula (I), the affinity substance is associated with the antibody, and then the reactive group covalently bound to the affinity substance via LB reacts with a group in the side chain of a specific amino acid residue present near the target site (e.g., an amino group in the side chain of a lysine residue), thereby covalently binding to the antibody. In this case, if the specific amino acid residue is not present in the vicinity of the specific amino acid residue to which R is to react and bind, the vicinity of the target site, and the region between the specific amino acid residue and the target site, the reactive group can selectively bind to the specific amino acid side chain position even if the length of the main chain is not strictly controlled. Of course, even when the specific amino acid residue is present elsewhere in such a region, the reactive group can be selectively bound to the specific amino acid residue position by controlling the length of the main chain.

[1010] The length of the main chain connecting A and R can be changed according to factors such as the type of antibody and affinity substance therefor, and the relationship between the position and number of specific amino acid residues in the target site in the antibody, and can be about above, preferably about Above, preferably about The length of such a main chain is, for example, about Below, preferably about Below, preferably about More specifically, the length of such a main chain is, for example, about It is better to Better to make an appointment

[1011] In addition, the relationship between the lengths (distances) of atoms is shown in the following table, which is common knowledge in the art. Therefore, those skilled in the art can use the lengths between atoms in the following table as a reference and appropriately design a backbone having the same length as the backbone described above. The corresponding number of atoms in the main chain.

[1012] [Table 1]

[1013] Table 1. Relationship between the lengths (distances) of carbon atoms at both ends of a linear alkylene group

[1014]

[1015] More specifically, the length of the main chain connecting A and R can also be determined according to the number of atoms constituting the main chain (excluding hydrogen atoms and substituents). The number of atoms constituting the main chain can be, for example, 4 (approximately ) or more, preferably 6 (about ) or more, preferably 8 (approximately ) or more. In addition, the number of atoms in the main chain can be, for example, 20 (approximately ) or less, preferably 16 (about ) or less, preferably 12 (approximately More specifically, the number of atoms in the main chain may be, for example, 4 to 20, preferably 6 to 16, and more preferably 8 to 12.

[1016] When the main chain has no ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure.

[1017] On the other hand, when the main chain is a structure containing a ring structure, the number of atoms in the main chain does not necessarily correspond to the above-mentioned length, and there is a tendency that the length specified by the number of atoms in the main chain is shorter than the above-mentioned length. Even in such a case, from the perspective of specifying the length of the main chain, the number of atoms in the main chain can be simply counted. Specifically, the number of atoms in the main chain in such a case can be determined by counting the number of atoms in the shortest path connecting the two bonds in the ring structure based on the number of atoms in the chain structure that does not contain a bivalent ring structure in the main chain (see, for example, the bold path in (a) to (d) below);

[1018] [Chemical Formula 34]

[1019]

[1020] ·Indicates a bond;

[1021] In the case of (a), the shortest path is the path in bold (boldface), so the number of atoms in the bivalent ring structure counted as the number of atoms in the main chain is 2;

[1022] In the case of (b), the shortest path is the bold path, so the number of atoms in the bivalent ring structure counted as the number of atoms in the main chain is 3;

[1023] In the case of (c), all paths are shortest paths (equal distances), so the number of atoms in the bivalent ring structure counted as the number of atoms in the main chain is 4;

[1024] In the case of (d), the path of the fused portion is the shortest path, and therefore the number of atoms in the bivalent ring structure counted as the number of atoms in the main chain is 4.

[1025] Preferably, the linking portion (excluding the side chain) between A and R represented by LB may be a chain structure without a bivalent ring structure. In this case, L and B may be appropriately designed so that the linking portion between A and R represented by LB does not contain a bivalent ring group.

[1026] 1-6-2. Specific structure of partial structure "LB"

[1027] In the above formula (I), L and B are structures that can be associated with each other. Therefore, in the above formula (I), L and B can be defined as a partial structure represented by "LB".

[1028] In one embodiment, the cleavable linker can be (i) a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side by cleavage, or (ii) a divalent group comprising a cleavable moiety not capable of generating a bioorthogonal functional group on the reactive group side by cleavage.

[1029] In a specific embodiment, when L is the cleavable linker of (i) above, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group.

[1030] Preferably, when L is the cleavable linker of (i) above, B is a divalent group (a) containing a bioorthogonal functional group. In this case, the bioorthogonal functional group generated in (i) and the bioorthogonal functional group in (a) may be of the same species or of different species. From the viewpoints of adopting a simpler structure and / or improving the reactivity to a single functional substance, the bioorthogonal functional group generated in (i) may be of the same species as the bioorthogonal functional group in (a). On the other hand, from the viewpoints of ensuring differentiated reactivity to more than two functional substances and not using a part of the bioorthogonal functional groups in the reaction, the bioorthogonal functional group generated in (i) may be of a different species from the bioorthogonal functional group in (a).

[1031] Alternatively, when L is the cleavable linker of (i) above, B may be (b) a divalent group that does not contain a bioorthogonal functional group. In this case, the compound represented by formula (I) or a salt thereof has a simpler structure and is therefore easier to synthesize.

[1032] In another specific embodiment, when L is the cleavable linker of (ii) above, B is (a) a divalent group containing a bioorthogonal functional group.

[1033] In a specific embodiment, the partial structure represented by LB preferably does not contain a peptide portion. In this case, the antibody having a functional substance of the present invention (e.g., an antibody-drug complex) obtained using the compound of the present invention has the advantage of not containing an immunogenic peptide portion as a linker.

[1034] In a specific embodiment, the partial structure represented by "LB" can have a symmetrical structure [e.g., cis (i.e., Z), trans (i.e., E)] based on the atom present at the center position of the main chain (the straight chain portion in LB) connecting A and R (e.g., when the number of atoms constituting the main chain is an odd number) or the binding site present at the center position of the main chain (e.g., when the number of atoms constituting the main chain is an even number). For example, the binding site present at the center position can also be designed as a cleavage moiety in the cleavage linker as described above. When the partial structure represented by "LB" has a symmetrical structure, the partial structure represented by "LB" can be easily synthesized. For example, a symmetrical structure (chain divalent group-SS-chain divalent group) containing a cleavage moiety at the center position of the main chain can be achieved by reacting the same divalent groups having a functional group capable of reacting to form a cleavage moiety at one end (e.g., a chain divalent group having an SH group at one end) with each other.

[1035] Therefore, the partial structure represented by "LB" can be a partial structure represented by "B2-L'-B1" (i.e., L is a divalent group represented by B2-L', and B is B1). In this case, the compound represented by the above formula (I) can be defined as a compound represented by the following formula (I');

[1036] A-B2-L'-B1-R(I')

[1037] Where,

[1038] A and R have the same meanings as the corresponding symbols in formula (I),

[1039] L' is a cleavable linker, which is a divalent group comprising a cleavable moiety, B1 and B2 are the same or different and are (a) a divalent group comprising a bioorthogonal functional group, or (b) a divalent group not comprising a bioorthogonal functional group,

[1040] B1 and B2 may have a symmetrical structure centered around L'.

[1041] B1 and B2 are the same as or different from each other, and have the same definitions, examples and preferred embodiments as B.

[1042] In a specific embodiment, L' can be represented by any one of the following formulas (L1') to (L3');

[1043] La'-C'-Lb' (L1')

[1044] La'-C' (L2')

[1045] C'-Lb' (L3')

[1046] Where,

[1047] La' and Lb' are divalent groups,

[1048] C' is the cutting part.

[1049] The divalent groups represented by La' and Lb' have the same definitions, examples and preferred examples as those of the divalent groups represented by La' and Lb', respectively.

[1050] The definition, examples and preferred examples of the cleavable moiety represented by C' are the same as those of the cleavable moiety represented by C.

[1051] In another specific embodiment, the structural unit represented by LB can be represented by the following formula (LB');

[1052] [Chemical Formula 35]

[1053]

[1054] Where,

[1055] C, p, p', q, q', X, X', R 1a 、R 1a' 、R 1b 、R 1b' , Y, Y', Z and Z' definitions, examples and preferred examples are the same as above,

[1056] ○ (white circle) indicates the bond to A, and ● (black circle) indicates the bond to R.

[1057] Therefore, the above formula (I) can be defined as the following formula (I”):

[1058] [Chemical Formula 36]

[1059]

[1060] Where,

[1061] A, R, C, p, p', q, q', X, X', R 1a 、R 1b 、R 1a' 、R 1b' The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above.

[1062] In the above formulas (LB') and (I"), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the same as the length of the main chain connecting A and R. In these formulas, the length from C in C=Z to C in C=Z' can also be specified as the number of atoms in the connecting chain (excluding hydrogen atoms and substituents) constituting the partial structure connecting C in C=Z to C in C=Z'. Such a number of atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain (excluding hydrogen atoms and substituents) that connects the partial structure connecting C in C=Z to C in C=Z' may or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[1063] 1-7. Manufacturing Method

[1064] A compound comprising an affinity substance for an antibody, a cleavable moiety, and a reactive group, or a salt thereof can be prepared appropriately. The compound comprising an affinity substance for an antibody, a cleavable moiety, and a reactive group is represented by formula (I), preferably formula (I'), and more preferably formula (I").

[1065] As an affinity substance, a substance with an arbitrary functional group can be appropriately selected. Therefore, by utilizing a reactive group that can react with the functional group, the affinity substance is reacted with a structural unit represented by LBR or a structural unit represented by RLBR (two reactive groups are the same or different), and a structural unit represented by ALBR can be prepared. For example, such a reaction can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous solution system at a suitable temperature (for example, about 15 to 200 ° C). The reaction system may include a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and further preferably 30 minutes to 8 hours.

[1066] In the reaction system, the molar ratio (Y / X) of the structural unit represented by LBR or the structural unit represented by RLBR to the affinity substance (X) varies depending on the types of the structural unit and the affinity substance, the number of sites in the affinity substance to be modified by the structural unit, etc., and is not particularly limited. For example, it is 0.1 to 50, preferably 0.5 to 40, more preferably 1 to 35, further preferably 2 to 25, and particularly preferably 3 to 15.

[1067] The formation of an antibody or a salt thereof containing an affinity substance for an antibody and a cleavable moiety can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although it depends on the specific starting materials and the molecular weight of the product. The antibody or a salt thereof containing an affinity substance for an antibody and a cleavable moiety can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[1068] 1-8. Others

[1069] In the inventions described later [e.g., the inventions represented by formulae (II) to (v) and their subordinate concepts and partial structural formulae (e.g., (L1) to (L3), (La'), (Lb'), (B-1) to (B-4))], arbitrary symbols (e.g., A, L, B, R), terms represented by these symbols, and their detailed descriptions (e.g., definitions, examples, and preferred examples) are common to the invention of the compound represented by the above-mentioned formula (I) or its salt. In addition, any technical elements (e.g., definitions, examples, and preferred examples) such as specific parts (e.g., cleavable parts, parts having the ability to generate bioorthogonal functional groups on the reactive group side by cleavage), specific groups (e.g., bioorthogonal functional groups, divalent groups, alkyl groups, substituents, electron-withdrawing groups), specific numerical values, and salts that can define the inventions described later may also be common to the contents described above. Therefore, unless otherwise specified, these matters may be appropriately cited in the inventions described later. Likewise, technical elements of specific inventions described in the inventions to be described later can be cited as technical elements of the present invention and other inventions as appropriate.

[1070] 2. Antibodies or their salts comprising an affinity substance for antibodies and a cleavable portion

[1071] 2-1. Overview

[1072] The present invention provides an antibody or a salt thereof represented by formula (II) comprising an affinity substance for the antibody and a cleavable portion;

[1073] ALB-R'-T (II)

[1074] Where,

[1075] A is an affinity substance for antibodies,

[1076] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[1077] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[1078] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1079] T stands for antibody.

[1080] 2-2. Moiety (R') generated by reaction between antibody and reactive group

[1081] In the portion generated by the reaction between the antibody and the reactive group, the definitions, examples, and preferred embodiments of the antibody and the reactive group are as described above. The portion generated by the reaction between the antibody and the reactive group is common knowledge in the art and can be appropriately determined according to the type of the antibody and the reactive group.

[1082] Preferably, the portion generated by the reaction between the antibody and the reactive group is a portion generated by the reaction between the side chain of any one of the 14 amino acids (asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine and lysine) that the antibody may contain and a reactive group thereof.

[1083] More preferably, the moiety generated by the reaction between the antibody and the reactive group may be a moiety generated by the reaction between the side chain of any one of lysine, tyrosine, tryptophan, and cysteine ​​and a reactive group specific thereto.

[1084] More preferably, the moiety generated by the reaction between the antibody and the reactive group can be a moiety generated by the reaction between the side chain of any one of the amino acids lysine, tyrosine, or tryptophan and a reactive group specific thereto. Examples of the moiety generated by the reaction between the side chain of any one of the amino acids lysine, tyrosine, or tryptophan and a reactive group specific thereto include the linking moieties and / or chemical structures described in "1-5. Reactive Group (R)" above.

[1085] Still more preferably, the moiety generated by the reaction between the antibody and the reactive group may be a moiety generated by the reaction between the side chain of lysine or tyrosine and a reactive group specific thereto (particularly when the antibody is human IgG1 or human IgG). Examples of the moiety generated by the reaction between the side chain of lysine or tyrosine and a reactive group specific thereto include the linking moieties and / or chemical structures described in "1-5. Reactive Group (R)" above.

[1086] Particularly preferably, the moiety generated by the reaction between the antibody and the reactive group may be a moiety generated by the reaction between the side chain of lysine and a reactive group specific thereto.

[1087] 2-3. Partial structure "LB"

[1088] The details of the partial structure "LB" are as described in the above "1-6. Partial structure 'LB'".

[1089] In a specific embodiment, the partial structure represented by "LB" may be a partial structure represented by "B2-L'-B1" (i.e., L is a divalent group represented by B2-L', and B is B1). In this case, the antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety represented by the above formula (II) may be represented by the following formula (II');

[1090] A-B2-L'-B1-R'-T (II')

[1091] Where,

[1092] A, R' and T have the same meanings as the corresponding symbols in formula (II),

[1093] L' is a cleavable linker, which is a divalent group comprising a cleavable moiety, B1 and B2 are the same or different and are (a) a divalent group comprising a bioorthogonal functional group, or (b) a divalent group not comprising a bioorthogonal functional group,

[1094] B1 and B2 may have a symmetrical structure centered around L'.

[1095] In the above formula (II'), L' can be represented by any one of the above formulas (L1') to (L3').

[1096] In another specific embodiment, the structural unit represented by LB can be represented by the following formula (LB');

[1097] [Chemical Formula 37]

[1098]

[1099] Where,

[1100] C, p, p', q, q', X, X', R 1a 、R 1a' 、R 1b 、R 1b' , Y, Y', Z and Z' definitions, examples and preferred examples are the same as above,

[1101] ○ (white circle) indicates the bond to A, and ● (black circle) indicates the bond to R'.

[1102] Therefore, the above formula (II) can be defined as the following formula (II”):

[1103] [Chemical Formula 38]

[1104]

[1105] Where,

[1106] A, R', T, C, p, p', q, q', X, X', R 1a 、R 1b 、R 1a' 、R 1b' The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above.

[1107] In the above formulas (LB') and (II"), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the same as the length of the main chain connecting A and R as described above. In these formulas, the length from C in C=Z to C in C=Z' can also be specified as the number of atoms in the connecting chain (excluding hydrogen atoms and substituents) constituting the partial structure connecting C in C=Z to C in C=Z'. Such a number of atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain (excluding hydrogen atoms and substituents) that connects the partial structure connecting C in C=Z to C in C=Z' may contain a ring structure or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[1108] 2-4. Binding Sites of Antibodies with Partial Structures Other Than Antibodies (Positional Selectivity)

[1109] A partial structure other than the antibody (eg, ALB-R') can positionally selectively bind to the target region of the antibody (T) as described above.

[1110] In this specification, "position-selective" or "position selectivity" means that although the specific amino acid residues in the antibody are not concentrated (biased) in a specific area, the specified structural unit that can bind to the specific amino acid residues in the antibody is concentrated in the specific area of ​​the antibody. Therefore, expressions related to position selectivity such as "position-selective possession", "position-selective binding", and "binding based on position selectivity" mean that "the retention rate or binding rate of the specified structural unit in the target area containing more than one specific amino acid residue" is significantly higher than "the retention rate or binding rate of the structural unit in the non-target area containing multiple amino acid residues of the same type as the specific amino acid residue in the target area". Such position-selective binding or retention can be achieved by the present invention, which can prevent the specified structural unit from reacting randomly with the specific amino acid residues in the antibody, but can preferentially react with the specific amino acid residues in the target area of ​​the antibody.

[1111] Specifically, when T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to the one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The definitions, examples, and preferred embodiments of the target region and positional selectivity are as described above.

[1112] 2-5. Number of partial structures other than antibodies possessed by the antibody

[1113] The number of partial structures other than the antibody (e.g., ALB-R') possessed by the antibody (T) can vary. For example, when T is a multimeric protein comprising a plurality of monomeric proteins, T may have partial structures other than T in a plurality of corresponding target regions in the plurality of monomeric proteins. Thus, T may have a plurality of partial structures other than T. Therefore, the structure represented by formula (II), (II') or (II") indicates that T may have one or more partial structures other than T. The number of partial structures other than T possessed by T can be adjusted by appropriately setting conditions such as the type of antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number also varies depending on the type of antibody and may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[1114] In a specific embodiment, the number of partial structures other than antibodies possessed by an antibody can be multiple if the antibody is a multimeric protein composed of multiple monomeric proteins. Using the present invention, multiple partial structures other than antibodies can be introduced into the same target region of multiple monomeric proteins.

[1115] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples and preferred embodiments of antibodies are as described above. The number of heavy chains varies depending on the type of antibody. For example, IgG, IgE and IgD may have 2 heavy chains. IgA may have 2 or 4 heavy chains. IgM may have 8 heavy chains. The number of partial structures other than antibodies retained by an antibody (antibody) may be considered as a parameter synonymous with DAR. In the present invention, the number of partial structures other than antibodies retained by an antibody may be 1 or 2 (preferably 2) for IgG, IgE and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[1116] 2-6. Manufacturing method

[1117] The present invention provides a method for producing an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety, comprising the following steps:

[1118] (A1) A compound comprising an affinity substance for an antibody, a cleavable moiety, and a reactive group, or a salt thereof, is reacted with an antibody to produce an antibody comprising an affinity substance for an antibody and a cleavable moiety, or a salt thereof.

[1119] The compound comprising an affinity substance for an antibody, a cleavable portion and a reactive group is represented by formula (I), preferably formula (I'), and even more preferably formula (I"). The antibody comprising an affinity substance for an antibody and a cleavable portion is represented by formula (II), preferably formula (II'), and even more preferably formula (II").

[1120] The compound or its salt comprising an affinity substance for an antibody, a cleavage portion and a reactive group has a reactive group and can therefore react with the antibody. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition (e.g., cleavage of an amide bond) of the protein. For example, such a reaction can be carried out in a suitable reaction system such as a buffer at room temperature (e.g., about 15 to 30° C.). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and further preferably 30 minutes to 8 hours. For details of such reactions, reference may be made to, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[1121] In the reaction system, the molar ratio (Y / X) of the compound containing an affinity substance for an antibody, a cleavable moiety, and a reactive group, or a salt thereof (Y) to the antibody (X) varies depending on the types of the compound containing an affinity substance for an antibody, a cleavable moiety, and a reactive group, and the antibody, the number of sites in the antibody to be modified by the compound containing an affinity substance for an antibody, a cleavable moiety, and a reactive group (e.g., DAR), and the like, and is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, further preferably 2 to 50, and particularly preferably 3 to 30.

[1122] The confirmation of the generation of an antibody or a salt thereof containing an affinity substance for an antibody and a cleavage portion, although it also depends on the specific molecular weight of the raw materials and the product, can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, preferably by mass spectrometry. The confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, it can be carried out, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As a protease, an intracellular protease is preferred. Examples of such intracellular proteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The confirmation of the number of partial structures other than the antibody possessed by the antibody can be carried out, for example, by electrophoresis, chromatography or mass spectrometry, preferably by mass spectrometry. The antibody or a salt thereof containing an affinity substance for an antibody and a cleavage portion can be appropriately purified by any method such as chromatography (e.g., the chromatography and affinity chromatography described above).

[1123] 3. Complexes of substances having affinity for antibodies, cleavable moieties, functional substances, and antibodies or their salts 3-1. Overview

[1124] The present invention provides a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, represented by the following formula (III);

[1125] AL-B'(-F)-R'-T (III)

[1126] Where,

[1127] A is an affinity substance for antibodies,

[1128] L is a cleavable linker, which is a divalent group containing a cleavable moiety,

[1129] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1130] F is a functional substance,

[1131] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1132] T stands for antibody.

[1133] In formula (III) or other formulas, R' is not F and is covalently bonded to B'.

[1134] 3-2. Functional substances (F)

[1135] As functional substance, as long as it is the substance that gives any function to antibody, then there is no particular limitation, for example, can be enumerated medicine, labeling substance, stabilizer, preferably medicine or labeling substance.In addition, functional substance can be single functional substance, or can be the substance that the functional substance of more than 2 is connected.

[1136] As medicine, it can be the medicine for any disease.As such disease, for example cancer (for example lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune disease and inflammatory disease (for example allergic disease, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve disease (for example cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious disease (for example bacterial infectious disease, viral infectious disease), hereditary disease and rare disease (for example hereditary spherocytosis, non-dystrophic myotonia syndrome), eye disease (for example age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), disease (for example deforming arthritis) in orthopedics and plastic surgery field, blood disease (for example leukemia, purpura), other diseases (for example metabolic abnormalities such as diabetes, hyperlipidemia, liver disease, kidney disease, lung disease, circulatory system disease, digestive system disease) can be listed. When the antibody is an antibody against a target protein of a predetermined disease, the drug may be a drug related to the predetermined disease (e.g., a drug for treating the predetermined disease, a drug for alleviating side effects associated with the use of the antibody against the target protein of the predetermined disease).

[1137] More specifically, the drug is an anticancer agent. Examples of anticancer agents include chemotherapeutics, toxins, radioactive isotopes, or substances containing the same. Examples of chemotherapeutics include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, microtubule binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin). Examples of radioactive isotopes include radioactive isotopes of hydrogen atoms (e.g., 3 H), radioactive isotopes of carbon atoms (such as 14 C), radioactive isotopes of phosphorus atoms (e.g. 32 P), radioactive isotopes of sulfur atoms (such as 35 S), radioactive isotopes of yttrium (e.g. 90 Y), radioactive isotopes of technetium (such as 99m Tc), radioactive isotopes of indium (such as 111 In), radioactive isotopes of iodine atoms (such as 123 I.125 I. 129 I. 131 I), radioactive isotopes of samarium (e.g. 153 Sm), radioactive isotopes of rhenium (e.g. 186 Re), radioactive isotopes of astatine (e.g. 211 At), radioactive isotopes of bismuth (such as 212 More specifically, the drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine, ), IGN, camptothecin analogs, calicheamicin, duocarmicin, eribulin, anthracycline drugs, dmDNA31, anti-tubulysin.

[1138] Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., fluorescein, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., the radioactive isotopes mentioned above), or substances containing the same.

[1139] In addition, the functional substance is a macromolecular compound, a medium molecular compound or a low molecular compound, preferably a low molecular compound. A low molecular compound refers to a compound with a molecular weight of 1500 or less. A low molecular compound is a natural compound or a synthetic compound. The molecular weight of the low molecular compound can be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less or 300 or less. In addition, the molecular weight of the low molecular compound can be more than 30, more than 40 or more than 50. The low molecular compound can be a drug or a labeling substance as described above. In addition, as the low molecular compound, for example, amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids and their salts can be listed.

[1140] 3-3. Trivalent Group (B') Containing a Moiety Generated by Reaction between a Functional Substance and a Bioorthogonal Functional Group: B' is the same as the divalent group containing a bioorthogonal functional group (a) in B described above, except that the bioorthogonal functional group in (a) reacts with the functional substance. Therefore, the definitions, examples, and preferred embodiments of the trivalent group and orthogonal functional group in B' are the same as those in B, except that the bioorthogonal functional group in (a) reacts with the functional substance.

[1141] Functional substances have various functional groups corresponding to their structures. When a functional substance has a functional group that is easy to react with a bioorthogonal functional group, the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group. The functional group that is easy to react with the bioorthogonal functional group can be different according to the specific type of the bioorthogonal functional group. If it is a person skilled in the art, an appropriate functional group can be appropriately selected as a functional group that is easy to react with the bioorthogonal functional group (for example, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). As a functional group that is easy to react with a bioorthogonal functional group, for example, when the bioorthogonal functional group is a thiol residue, maleimide residues and disulfide residues can be listed, when the bioorthogonal functional group is an alkyne residue, azide residues can be listed, and when the bioorthogonal functional group is an aldehyde residue or a ketone residue, hydrazine residues can be listed, but are not limited to these. For example, when the bioorthogonal functional group is a thiol residue and the functional group that readily reacts with the bioorthogonal functional group is a maleimide residue or a disulfide residue (or vice versa), the trivalent group containing the portion generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a sulfosuccinimide residue or a trivalent group containing a disulfide residue; when the bioorthogonal functional group is an alkyne residue and the functional group that readily reacts with the bioorthogonal functional group is an azide residue (or vice versa), the trivalent group containing the portion generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a sulfosuccinimide residue or a trivalent group containing a disulfide residue. The trivalent group containing a portion generated by the reaction between the functional group and the bioorthogonal functional group may be a trivalent group containing a triazole residue (which may or may not be fused with other rings). When the bioorthogonal functional group is an aldehyde residue or a ketone residue and the functional group that readily reacts with the bioorthogonal functional group is a hydrazine residue (or vice versa), the trivalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a hydrazone residue (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Preferred examples of the trivalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group include a trivalent group containing a thiosuccinimide residue, a trivalent group containing a disulfide residue, a trivalent group containing a triazole residue (which may or may not be fused with other rings), or a trivalent group containing a hydrazone residue.

[1142] On the other hand, when the functional substance does not have a functional group that readily reacts with a bioorthogonal functional group, a substance that has been derivatized to have the desired functional group can be used as the functional substance. For example, when the functional substance is an antibody, a substance that has been derivatized to have a functional group that the antibody does not naturally have can be used. In this case, the antibody can be derivatized using the method of the present invention. In this case, the antibody that has been derivatized to selectively have the desired bioorthogonal functional group can be used as the functional substance.

[1143] Derivatization is common technical knowledge in this field (e.g., International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, and U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization can be performed using a cross-linking agent as described above. Alternatively, derivatization can be performed using a specific linker having a desired functional group. For example, such a linker can be one that can separate the functional substance from the antibody by cleavage of the linker in an appropriate environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers that are degraded by specific proteases [e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory proteases)] (e.g., U.S. Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83: 67-123 (1999)), linkers that can be cleaved at local acidic sites present in the body (e.g., U.S. Patent No. 5,622,929, U.S. Patent No. 5,122,368, U.S. Patent No. 5,824,805). The linker may be self-immolative (e.g., International Publication No. 02 / 083180, International Publication No. 04 / 043493, International Publication No. 05 / 112919). In the present invention, the derivatized functional substance may also be simply referred to as a "functional substance."

[1144] In trivalent groups comprising a moiety generated by the reaction between a functional substance and a bioorthogonal functional group, the definitions, examples, and preferred embodiments of the functional substance and bioorthogonal functional group are as described above. The moiety generated by the reaction between a functional substance and a bioorthogonal functional group is common knowledge in the art and can be appropriately determined based on the types of the functional substance (or, if the functional substance is derivatized, the derivatized moiety) and the bioorthogonal functional group.

[1145] The trivalent group comprising a moiety generated by the reaction between a functional substance and a bioorthogonal functional group may be (1) a trivalent group comprising a thiosuccinimide residue, a triazole residue, or a hydrazone residue as the residues mentioned in the preferred examples above, or (2) a trivalent group comprising a residue selected from, for example, a disulfide residue (the residue is the residue mentioned in the preferred examples above), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue, but is not particularly limited.

[1146] Furthermore, the trivalent group comprising a moiety generated by the reaction between the functional substance and the bioorthogonal functional group is not particularly limited, and for example, may be a trivalent group comprising a residue corresponding to any one of the chemical structures selected from the following group;

[1147] [Chemical Formula 39]

[1148]

[1149] Here,

[1150] The black squares represent the bonds to the functional substance side.

[1151] ○ (white circle) represents the bond to the L-side portion, and ● (black circle) represents the bond to the R'-side portion. When the chemical structure is asymmetric around the cleavable portion, ● may represent the bond to the L-side portion, and ○ may represent the bond to the R'-side portion.

[1152] 3-4. Partial structure "L-B'(-F)"

[1153] The details of the partial structure "LB'(-F)" are the same as those described in the above "1-6. Partial structure 'LB'" except that B is changed to B'(-F).

[1154] In one embodiment, the cleavable linker can be (i) a divalent group comprising a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side by cleavage, or (ii) a divalent group comprising a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side by cleavage.

[1155] In a specific embodiment, the cleavable linker may be the cleavable linker described in (ii) above. This is because since B' is already bound to the functional substance (F), there is no need to generate a bioorthogonal functional group on the reactive group side.

[1156] In another specific embodiment, the partial structure represented by L-B'(-F) may be a partial structure represented by B2'(-F2)-L'-B1'(-F1)" (i.e., L is B2'(-F2)-L', and B' is B1'). In this case, the complex of the substance having affinity for the antibody, the cleavage moiety, the functional substance, and the antibody represented by the above formula (III), or a salt thereof, may be represented by the following formula (III');

[1157] A-B2'(-F2)-L'-B1'(-F1)-R'-T (III')

[1158] Where,

[1159] A, R' and T have the same meanings as the corresponding symbols in formula (III),

[1160] L' is a cleavable linker, which is a divalent group containing a cleavable portion, B1' and B2' are the same or different and are a trivalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1161] F1 and F2 are the same or different and are functional substances.

[1162] B1'(-F1) and B2'(-F2) may have a symmetrical structure centered around L'.

[1163] B1' and B2' are the same or different, and have the same definitions, examples and preferred embodiments as B'.

[1164] In the above formula (III'), L' can be represented by any one of the above formulas (L1') to (L3').

[1165] In another specific embodiment, the structural unit represented by LB'(-F) can be represented by the following formula (LB'(F)');

[1166] [Chemical Formula 40]

[1167]

[1168] Where,

[1169] C, F1, F2, p, p', q, q', X, X', R 1a 、R 1a' 、R 1b 、R 1b', Z and Z' definitions, examples and preferred examples are the same as above,

[1170] Y and Y' are the same or different and are residues obtained by removing one hydrogen atom from Y of the formula (B-1),

[1171] ○ (white circle) indicates the bond to A, and ● (black circle) indicates the bond to R'.

[1172] More specifically, the residue obtained by removing one hydrogen atom from Y in formula (B-1) in Y and Y' is -N(-)-, -CH(-)-, or a group obtained by removing one hydrogen atom from formula (B-2). From the perspective of structural simplification, Y may be -N(-)- or -CH(-)-. Alternatively, from the perspective of designing a structure based on carbon atoms, Y may be -CH(-)- or a group obtained by removing one hydrogen atom from formula (B-2).

[1173] The group obtained by removing one hydrogen atom from the above formula (B-2) is a group obtained by removing one hydrogen atom from the following formula (B-2');

[1174] [Chemical Formula 41]

[1175]

[1176] Where,

[1177] V and V' are the same or different and are -NH-, -O-, -CH2- or a single bond,

[1178] V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[1179] s is an arbitrary integer from 0 to 10, and ○ and ● in formula (B-2') have the same orientations as ○ and ● in formula (B-1), respectively. The definition, examples, and preferred embodiments of s in formula (B-2') are the same as those in formula (B-2). Therefore, in formula (B-2'), one of V and V' may be -N(-)- or -CH(-)-. Alternatively, in formula (B-2'), V1 may be a trivalent group obtained by removing one hydrogen atom from the divalent group of (a) or (b) above. Alternatively, in formula (B-2'), one of the s -CH2- may be -CH(-)-.

[1180] In this case, the above formula (III) can be defined as the following formula (III”):

[1181] [Chemical Formula 42]

[1182]

[1183] Where,

[1184] A, R, C, F1, F2, p, p', q, q', X, X', R 1a 、R 1b 、R 1a' 、R 1b' The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above.

[1185] In the above formulas (LB'(F)') and (III"), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the same as the length of the main chain connecting A and R as described above. In these formulas, the length from C in C=Z to C in C=Z' can also be specified as the number of atoms in the connecting chain (excluding hydrogen atoms and substituents) constituting the partial structure connecting C in C=Z to C in C=Z'. Such a number of atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain (excluding hydrogen atoms and substituents) that connects the partial structure connecting C in C=Z to C in C=Z' may contain a ring structure or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[1186] 3-5. Binding Sites of Antibodies with Partial Structures Other Than Antibodies (Positional Selectivity)

[1187] The partial structure other than the antibody (e.g., AL-B'(-F)-R') can positionally selectively bind to the target region described above in the antibody (T). Specifically, when T contains one or more specific amino acid residues in a target region formed by 1 to 50 consecutive amino acid residues and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The definitions, examples, and preferred embodiments of the target region and positional selectivity are as described above.

[1188] 3-6. Number of partial structures other than antibodies possessed by the antibody

[1189] The number of partial structures other than the antibody (e.g., AL-B'(-F)-R') possessed by the antibody (T) may vary. For example, when T is a multimeric protein comprising a plurality of monomeric proteins, T may have partial structures other than T in a plurality of corresponding target regions in the plurality of monomeric proteins. Thus, T may have a plurality of partial structures other than T. Therefore, the structure represented by formula (III), (III') or (III") indicates that T may have one or more partial structures other than T. The number of partial structures other than T possessed by T can be adjusted by appropriately setting conditions such as the type of antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number also varies depending on the type of antibody and may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[1190] In a specific embodiment, when the antibody is a multimeric protein composed of multiple monomeric proteins, the number of partial structures other than antibodies retained by the antibody may be multiple. If the present invention is used, multiple partial structures other than antibodies can be introduced into the same target region of multiple monomeric proteins.

[1191] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred embodiments of antibodies are as described above. In the present invention, the number of partial structures other than antibodies retained by an antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[1192] 3-7. Manufacturing method

[1193] The present invention provides a method for producing a complex of a substance having affinity for an antibody, a cleavage moiety, a functional substance, and an antibody, or a salt thereof, comprising the following steps:

[1194] (B1) reacting an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety with a functional substance to generate a complex comprising the affinity substance for the antibody, the cleavage moiety, the functional substance, and the antibody or a salt thereof.

[1195] An antibody comprising an affinity substance for an antibody and a cleavage portion is represented by formula (II), preferably formula (II'), and even better formula (II"). A complex comprising an affinity substance for an antibody, a cleavage portion, a functional substance and an antibody is represented by formula (III), preferably formula (III'), and even better formula (III").

[1196] Antibodies or their salts containing an affinity substance for the antibody and a cleavable moiety have bioorthogonal functional groups that react with functional substances and can therefore react with functional substances. Such reactions can be suitably carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., amide bond cleavage) as described in "2-6. Production Methods" above.

[1197] In the reaction system, the molar ratio (Y / X) of the "functional substance (Y)" to the "antibody or its salt (X) comprising an affinity substance for the antibody and a cleavage portion" varies depending on the types of the antibody and the functional substance, the number of sites in the antibody to be modified by the functional substance (e.g., DAR), etc., and is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, further preferably 2 to 50, and particularly preferably 3 to 30.

[1198] The formation of a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although this depends on the specific starting materials and product molecular weights. Confirmation of positional selectivity, confirmation of the number of partial structures other than antibodies, and purification can be appropriately performed by the methods described in "2-6. Production Methods" above.

[1199] The present invention also provides a method for producing a complex of a substance having affinity for an antibody, a cleavage moiety, a functional substance, and an antibody, or a salt thereof, comprising the following steps:

[1200] (B2) reacting a compound comprising an affinity substance for the antibody, a cleavable moiety, and a reactive group, or a salt thereof, with the antibody to produce the antibody comprising the affinity substance for the antibody and the cleavable moiety, or a salt thereof; and

[1201] (B3) reacting an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety with a functional substance to generate a complex comprising the affinity substance for the antibody, the cleavage moiety, the functional substance, and the antibody or a salt thereof.

[1202] The above-mentioned process (B2) can be carried out in the same manner as the above-mentioned process (A1) described in the above-mentioned "2-6. Manufacturing method". The above-mentioned process (B3) can be carried out in the same manner as the above-mentioned process (B1). The above-mentioned processes (B2) and (B3) can be carried out separately. Alternatively, the above-mentioned processes (B2) and (B3) can be carried out simultaneously based on the combination of the reaction pair of the specific amino acid residue and the reactive group that is the reaction target in the antibody, and the reaction pair of the functional group in the functional substance and the bioorthogonal functional group. That is, when the above-mentioned processes (B2) and (B3) are carried out simultaneously, the product obtained in the above-mentioned process (B2) can be provided to the above-mentioned process (B3) without separating the product obtained in the above-mentioned process (B2).

[1203] 4. Antibodies or their salts having bioorthogonal functional groups

[1204] 4-1. Overview of Manufacturing Method

[1205] The present invention provides a method for producing an antibody or a salt thereof having a bioorthogonal functional group represented by formula (IV);

[1206] L1-B-R'-T (IV)

[1207] Where,

[1208] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group,

[1209] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[1210] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1211] T stands for antibody.

[1212] 4-2. (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group (L1) not containing a bioorthogonal functional group

[1213] In formula (IV), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. Such a monovalent group is a monovalent group that can be generated by cleavage of a cleavable linker that is a divalent group containing a cleavable moiety as described above. Therefore, L1 can be a monovalent group that can be generated by cleavage of the residue or chemical structure of the cleavable moiety as described above. More specifically, (i') a monovalent group containing a bioorthogonal functional group can be a monovalent group that can be generated by cleavage of the above-mentioned "(i) a cleavable linker that is a divalent group containing a cleavable moiety that has the ability to generate a bioorthogonal functional group on the reactive group side by cleavage", and (ii') a monovalent group that does not contain a bioorthogonal functional group can be a monovalent group that can be generated by cleavage of "(ii) a cleavable linker that is a divalent group containing a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side by cleavage".

[1214] In a specific embodiment, L1 can be represented by any of the following formulas (L1-1) or (L1-2);

[1215] C1-Lb (L1-1)

[1216] C1 (L1-2)

[1217] Where,

[1218] Lb is a divalent group,

[1219] C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group.

[1220] Examples of the divalent group include a divalent hydrocarbon group which may have a substituent, a divalent heterocyclic group which may have a substituent, -C(=O)-, -NR a -(R a represents a hydrogen atom or a substituent), -O-, -S-, -C(═S)-, and a group composed of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of these. The definitions, examples, and preferred examples of the divalent hydrocarbon group, divalent heterocyclic group, and substituent are as described above.

[1221] The definition, examples and preferred examples of the bioorthogonal functional group are as described above.

[1222] As groups other than bioorthogonal functional groups, for example, groups other than bioorthogonal functional groups in the above-mentioned substituents can be cited. As such groups other than bioorthogonal functional groups, for example, alkyl, cycloalkyl, aralkyl, monovalent heterocyclic groups, hydroxyl, amino, alkyloxy (alkoxy), cycloalkyloxy, and aralkyloxy groups can be cited. Definitions, examples, and preferred examples of these groups and the constituent elements of these groups (for example, alkyl in alkyloxy (alkoxy), cycloalkyl in cycloalkyloxy, and aralkyl in aralkyloxy) are the same as those described above.

[1223] In a specific embodiment, Lb can be represented by the following formula (Lb');

[1224] [Chemical Formula 43]

[1225]

[1226] Where,

[1227] p is an arbitrary integer from 0 to 10,

[1228] q is an arbitrary integer from 0 to 10,

[1229] X is a carbon atom, a nitrogen atom or a single bond (in the case where X is a nitrogen atom, R 1b does not exist; when X is a single bond, R 1a and R 1b does not exist),

[1230] R 1a and R 1b are the same or different and are selected from hydrogen atoms or the above-mentioned substituents,

[1231] ○ (white circle) indicates the bond to C1, and ● (black circle) indicates the bond to B.

[1232] p, q, X, and R 1a and R 1b The definitions, examples and preferred examples of (substituent) are as described above.

[1233] 4-3. Partial structure "L1-B"

[1234] 4-3-1. Length of the partial structure "L1-B" connecting the end of L1 and R'

[1235] In formula (IV), the length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage (the straight chain portion in "L1-B") (or, not limited to specific formulas such as formula (IV), the number of atoms in the main chain connecting the "bioorthogonal functional group" and the "side chain of a specific amino acid residue" in an antibody or a salt thereof that selectively has a bioorthogonal functional group, the same below) can be appropriately designed based on various factors as described in the above "1-6-1. Length of the main chain in the partial structure 'L-B' connecting A and R".

[1236] The length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage can be, for example, about above, preferably about Above, preferably about The length of the partial structure can be, for example, about Below, preferably about Below, preferably about More specifically, the length of the partial structure can be, for example, about It is better to Better to make an appointment

[1237] More specifically, the length of the partial structure connecting the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage can also be specified as the number of atoms (excluding hydrogen atoms and substituents) constituting the connecting chain of the partial structure. The number of atoms constituting the connecting chain can be, for example, 2 (approximately ) or more, preferably 3 (about ) or more, preferably 4 (approximately ) or more. In addition, the number of atoms constituting the connecting chain may be, for example, 10 (approximately ) or less, preferably 8 (about ) or less, preferably 6 (approximately More specifically, the number of atoms constituting the linking chain may be, for example, 2 to 10, preferably 3 to 8, and more preferably 4 to 6.

[1238] When the linking chain connecting the L1 terminal portion (or C1 terminal portion) and the R' partial structure generated by cleavage is a chain structure without a bivalent ring structure, the number of atoms in the linking chain can be determined by counting the number of atoms in the linking chain.

[1239] On the other hand, when the connecting chain connecting the L1 terminal portion (or C1 terminal portion) and the partial structure of R' generated by cleavage is a structure containing a bivalent ring structure, the number of atoms in the connecting chain does not necessarily correspond to the above-mentioned length, and there is a tendency that the length specified by the number of atoms in the connecting chain is shorter than the above-mentioned length. Even in such a case, from the perspective of specifying the length of the connecting chain by the number of atoms, the number of atoms in the connecting chain can be simply counted. Specifically, the number of atoms in the connecting chain in such a case can be determined by counting the number of atoms in the shortest path connecting the two bonding bonds in the bivalent ring structure on the basis of the number of atoms in the chain structure that does not contain a bivalent ring structure in the connecting chain as described above.

[1240] Preferably, the linking chain connecting the L1 terminal portion (or C1 terminal portion) and the R' partial structure generated by cleavage may be a chain structure that does not contain a bivalent ring structure. In this case, L1-B can be appropriately designed so that the linking chain connecting the terminal portion generated by cleavage and the R' partial structure does not contain a bivalent ring group.

[1241] In a specific embodiment, the partial structure represented by L1-B preferably does not contain a peptide portion. In this case, the antibody (e.g., antibody-drug complex) having a functional substance of the present invention obtained using the antibody having a bioorthogonal functional group of the present invention has the advantage of not containing an immunogenic peptide portion as a linker.

[1242] 4-3-2. Specific structure of partial structure "L1-B"

[1243] In the above formula (IV), L1 and B are structures that can be associated with each other. Therefore, in the above formula (IV), L1 and B can be defined as a partial structure represented by "L1-B".

[1244] In a specific embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group.

[1245] Preferably, when L1 is a monovalent group (i') containing a bioorthogonal functional group, B is a divalent group (a) containing a bioorthogonal functional group. In this case, the bioorthogonal functional group in (i') and the bioorthogonal functional group in (a) may be of the same species or of different species. From the viewpoint of adopting a simpler structure and / or improving the reactivity to a single functional substance, the bioorthogonal functional group in (i') may be of the same species as the bioorthogonal functional group in (a). On the other hand, from the viewpoint of ensuring differentiated reactivity to more than two functional substances and not using some of the bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i') may be of a different species from the bioorthogonal functional group in (a).

[1246] Alternatively, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B can be (b) a divalent group not containing a bioorthogonal functional group. In this case, the antibody or salt thereof having a bioorthogonal functional group represented by formula (IV) has a simpler structure and is therefore easier to synthesize.

[1247] In another specific embodiment, when L1 is (ii') a monovalent group not containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group.

[1248] In a specific embodiment, the structural unit represented by L1-B can be represented by the following formula (L1B');

[1249] [Chemical Formula 44]

[1250]

[1251] Where,

[1252] C1, p, q, X, R 1a 、R 1b , the definitions, examples and preferred examples of Y and Z are the same as those described above,

[1253] ●(black circle) represents the bond to R'.

[1254] Therefore, the above formula (IV) can be defined as the following formula (IV');

[1255] [Chemical Formula 45]

[1256]

[1257] Where,

[1258] C1, p, q, X, R 1a 、R 1b The definitions, examples and preferred examples of R, Y, Z, R' and T are the same as those described above.

[1259] In the above formulas (L1B') and (IV'), the length from C to C1 in C=Z is the same as the length of the partial structure connecting the L1 terminal part (or C1 terminal part) and R' generated by cleavage. In these formulas, the length from C to C1 in C=Z can also be specified according to the number of atoms in the connecting chain (excluding hydrogen atoms and substituents) constituting the partial structure from C to C1 in C=Z. Such a number of atoms is the same as the number of atoms constituting "the partial structure (excluding hydrogen atoms and substituents) connecting the L1 terminal part (or C1 terminal part) and R' generated by cleavage". The connecting chain (excluding hydrogen atoms and substituents) connecting the partial structure from C to C1 in C=Z may contain a ring structure or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[1260] 4-4. Binding Sites of Antibodies with Partial Structures Other Than Antibodies (Positional Selectivity)

[1261] The partial structure other than the antibody (e.g., L1-B-R') can positionally selectively bind to the target region described above in the antibody (T). Specifically, when T contains one or more specific amino acid residues in a target region formed by 1 to 50 consecutive amino acid residues and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The definitions, examples, and preferred embodiments of the target region and positional selectivity are as described above.

[1262] 4-5. Number of partial structures other than antibodies possessed by the antibody

[1263] The number of partial structures other than the antibody (e.g., L1-B-R') possessed by the antibody (T) may vary. For example, when T is a multimeric protein comprising a plurality of monomeric proteins, T may have partial structures other than T in a plurality of corresponding target regions in the plurality of monomeric proteins. Thus, T may have a plurality of partial structures other than T. Therefore, the structure represented by formula (IV), (IV') or (IV") indicates that T may have one or more partial structures other than T. The number of partial structures other than T possessed by T can be adjusted by appropriately setting conditions such as the type of antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number also varies depending on the type of antibody and may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[1264] In a specific embodiment, the number of partial structures other than antibodies possessed by an antibody can be multiple if the antibody is a multimeric protein composed of multiple monomeric proteins. Using the present invention, multiple partial structures other than antibodies can be introduced into the same target region of multiple monomeric proteins.

[1265] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred embodiments of antibodies are as described above. In the present invention, the number of partial structures other than antibodies retained by an antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[1266] 4-6. Manufacturing method

[1267] The present invention provides a method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising the following steps:

[1268] (C1) Cleavage of the cleavable portion of an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavable portion to generate an antibody or a salt thereof having a bioorthogonal functional group.

[1269] An antibody comprising an affinity substance for an antibody and a cleavage portion is represented by formula (II), preferably formula (II'), and even more preferably formula (III"). An antibody having a bioorthogonal functional group is represented by formula (IV), preferably formula (IV'), and even more preferably formula (IV").

[1270] The antibody or its salt containing an affinity substance for the antibody and a cleavage moiety has a cleavage moiety that can be cleaved by the cleavage treatment described above, and thus can be cleaved. Such a cleavage reaction can be suitably carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds), as described in "2-6. Production Methods" above.

[1271] Examples of cleavage treatments include: (a) treatment with one or more substances selected from acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with a physicochemical stimulus selected from light; or (c) placement using a cleavage linker containing a self-degradable cleavage portion. The conditions for such cleavage treatment are common technical knowledge in this field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society. 132, 17928 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)).

[1272] The formation of an antibody or salt thereof having a bioorthogonal functional group can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although this depends on the specific starting materials and the molecular weight of the product. Confirmation of positional selectivity, confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described in "2-6. Production Methods" above.

[1273] The present invention also provides a method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising the following steps:

[1274] (C2) reacting a compound comprising an affinity substance for the antibody, a cleavable moiety, and a reactive group, or a salt thereof, with the antibody to produce the antibody comprising the affinity substance for the antibody and the cleavable moiety, or a salt thereof; and

[1275] (C3) cleaving the cleavage portion of the antibody or its salt comprising an affinity substance for the antibody and a cleavage portion to generate an antibody or its salt having a bioorthogonal functional group.

[1276] The above-mentioned process (C2) can be carried out in the same manner as the above-mentioned process (A1) described in the above-mentioned "2-6. Manufacturing method". The above-mentioned process (C3) can be carried out in the same manner as the above-mentioned process (C1). The above-mentioned processes (C2) and (C3) can be carried out separately. Alternatively, the above-mentioned processes (C2) and (C3) can be carried out simultaneously depending on factors such as the combination of the reactive group and the bioorthogonal functional group that can be generated by cleavage (non-reactive). That is, when the above-mentioned processes (C2) and (C3) are carried out simultaneously, the product obtained in the above-mentioned process (C2) can be provided to the above-mentioned process (C3) without separating the product obtained in the above-mentioned process (C2).

[1277] 5. Method for producing an antibody or a salt thereof having a functional substance

[1278] 5-1. Summary

[1279] The present invention provides a method for producing an antibody or a salt thereof having a functional substance represented by formula (V);

[1280] F-(L1-B)'-R'-T (V)

[1281] Where,

[1282] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group,

[1283] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, and the structural unit represented by (L1-B)' is a divalent structural unit containing a portion generated by the reaction between the "functional substance" and "either or both of the bioorthogonal functional groups in (i') and (a)",

[1284] F is a functional substance,

[1285] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1286] T stands for antibody.

[1287] 5-2. Partial structure "F-(L1-B)'" 5-2-1. Length of the partial structure "L1-B" connecting F and R'

[1288] In formula (V), the length of the partial structure "L1-B" connecting F and R' (the straight chain portion in "L1-B") (or, not limited to specific formulas such as formula (V), the number of atoms in the main chain connecting the "functional substance" and the "side chain of a specific amino acid residue" in an antibody or a salt thereof that selectively possesses a functional substance, or the number of atoms in the main chain connecting the "bioorthogonal functional group" and the "side chain of a specific amino acid residue" when the functional substance is bound to the antibody via a bioorthogonal functional group. The same applies hereinafter) can be appropriately designed based on various factors as described in the above "1-6-1. Length of the main chain in the partial structure 'L-B' connecting A and R".

[1289] The length of the partial structure connecting F and R' is, for example, about above, preferably about Above, preferably about The length of the partial structure is, for example, about Below, preferably about Below, preferably about More specifically, the length of the partial structure is, for example, about It is better to Better to make an appointment

[1290] More specifically, the length of the partial structure connecting F and R' can also be determined according to the number of atoms (excluding hydrogen atoms and substituents) that constitute the partial structure. The number of atoms that constitute the connecting chain can be, for example, 2 (approximately ) or more, preferably 3 (about ) or more, preferably 4 (approximately The number of atoms constituting the connecting chain can be, for example, 10 (approximately ) or less, preferably 8 (about ) or less, preferably 6 (approximately More specifically, the number of atoms constituting the linking chain may be, for example, 2 to 10, preferably 3 to 8, and more preferably 4 to 6.

[1291] When the connecting chain of the partial structure connecting F and R' is a chain structure not containing a bivalent ring structure, the number of atoms in the connecting chain can be determined by counting the number of atoms in the connecting chain.

[1292] On the other hand, when the connecting chain connecting the partial structure of F and R' is a structure containing a bivalent ring structure, the number of atoms in the connecting chain does not necessarily correspond to the above-mentioned length, and there is a tendency that the length specified by the number of atoms in the connecting chain is shorter than the above-mentioned length. Even in such a case, from the perspective of specifying the length of the connecting chain by the number of atoms, the number of atoms in the connecting chain can be simply counted. Specifically, the number of atoms in the connecting chain in such a case can be determined by counting the number of atoms in the shortest path connecting the two bonds in the bivalent ring structure, based on the number of atoms in the chain structure that does not contain a bivalent ring structure in the connecting chain, as described above.

[1293] Preferably, the connecting chain connecting the partial structure of F and R' may be a chain structure without a bivalent ring structure. L1-B may be appropriately designed so that the connecting chain connecting the partial structure of F and R' does not contain a bivalent ring group.

[1294] In a specific embodiment, the partial structure represented by L1-B preferably does not contain a peptide portion. In this case, the antibody (eg, antibody-drug complex) containing a functional substance of the present invention has the advantage of not containing an immunogenic peptide portion as a linker.

[1295] 5-2-2. Specific structure of partial structure "F-(L1-B)'"

[1296] In formula (V), L1 and B in F-(L1-B)' are structures that can be associated with each other. Therefore, in the above formula (V), L1 and B can be defined as a partial structure represented by "L1-B".

[1297] In addition, the structural unit represented by F-(L1-B)' is a portion generated by the reaction between the "functional substance" and "either one or both of the bioorthogonal functional groups in (i') and (a)". The bioorthogonal functional group provided for the reaction does not exist in formula (V). Therefore, in formula (V), neither one or both of the bioorthogonal functional groups in (i') and (a) are present. The portion generated by the reaction between the "functional substance" and "either one or both of the bioorthogonal functional groups in (i') and (a)" is the same as the portion generated by the reaction between the functional substance and the bioorthogonal functional group. Therefore, in this specification, the examples (names of the residues) and specific examples (chemical structural formulas) of the portion generated by the reaction between the "functional substance" and "either one or both of the bioorthogonal functional groups in (i') and (a)" are the same as those described above with respect to the portion generated by the reaction between the functional substance and the bioorthogonal functional group.

[1298] In one embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group.

[1299] In a preferred embodiment, when L1 is a monovalent group (i') containing a bioorthogonal functional group, B is a divalent group (a) containing a bioorthogonal functional group. In this case, the bioorthogonal functional group in (i') and the bioorthogonal functional group in (a) may be of the same species or of different species. From the viewpoints of adopting a simpler structure and / or improving the reactivity for a single functional substance, the bioorthogonal functional group in (i') may be of the same species as the bioorthogonal functional group in (a). On the other hand, from the viewpoints of ensuring differentiated reactivity for more than two functional substances and not using a part of the bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i') may be of a different species than the bioorthogonal functional group in (a).

[1300] In another preferred embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B can be (b) a divalent group not containing a bioorthogonal functional group. In this case, the antibody or salt thereof having a functional substance represented by formula (V) has a simpler structure and is therefore easier to synthesize.

[1301] In another embodiment, when L1 is (ii') a monovalent group not containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group.

[1302] In a specific embodiment, when the bioorthogonal functional groups in (i') and (a) are of different types, the antibody or a salt thereof having a functional substance represented by formula (V) can be represented by formula (V1) or (V2).

[1303] L1-B'(-F)-R'-T (V1)

[1304] Where,

[1305] L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group,

[1306] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1307] F is a functional substance,

[1308] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1309] T stands for antibody.

[1310] F-L1'-B-R'-T (V2)

[1311] Where,

[1312] L1′ is a divalent group including a portion generated by the reaction between the “functional substance” and the “(i′) monovalent group including a bioorthogonal functional group”,

[1313] B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group,

[1314] F is a functional substance,

[1315] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1316] T stands for antibody.

[1317] L1' is a divalent group containing a moiety generated by the reaction between the "functional substance" and the "(i') monovalent group containing a bioorthogonal functional group." The moiety generated by the reaction between the "functional substance" and the "(i') monovalent group containing a bioorthogonal functional group" is the same as the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. Therefore, in this specification, examples (residue names) and specific examples (chemical structural formulas) of the moiety generated by the reaction between the "functional substance" and the "(i') monovalent group containing a bioorthogonal functional group" are the same as those described above for the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. Therefore, since the functional substance reacts with the bioorthogonal functional group, the divalent group (L1') containing a portion generated by the reaction between the "functional substance" and the "(i') monovalent group containing a bioorthogonal functional group" can also be expressed as a divalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group (the same below). More specifically, regarding the divalent group containing a portion generated by the reaction between the functional substance and the bioorthogonal functional group, (1) it may be a divalent group containing a thiosuccinimide residue, a triazole residue, or a hydrazone residue as the residue mentioned in the preferred examples above, or (2) it may be a divalent group containing a residue selected from, for example, a disulfide residue (the residue is the residue mentioned in the preferred examples above), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue, but is not particularly limited; preferably, (1) it is a divalent group containing a thiosuccinimide residue, a triazole residue, or a hydrazone residue.

[1318] In another specific embodiment, when the bioorthogonal functional groups in (i') and (a) are of the same or different species, the antibody or a salt thereof having a functional substance represented by formula (V) can be represented by formula (V3).

[1319] Fa-L1'-B'(-Fb)-R'-T (V3)

[1320] Where,

[1321] L1′ is a divalent group including a portion generated by the reaction between the “functional substance” and the “(i′) monovalent group including a bioorthogonal functional group”,

[1322] B' is a trivalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1323] Fa and Fb are the same or different functional substances,

[1324] R' is a moiety generated by the reaction between the antibody and the reactive group,

[1325] T stands for antibody.

[1326] In a specific embodiment, in the above formula (V1), the structural unit represented by L1-B'(-F) can be represented by the following formula (L1B'(F));

[1327] [Chemical Formula 46]

[1328]

[1329] Where,

[1330] F, C1, p, q, X, R 1a 、R 1b The definitions, examples and preferred examples of , and Z are the same as those described above,

[1331] Y' is a residue obtained by removing one hydrogen atom from Y of the formula (B-1),

[1332] ●(black circle) represents the bond to R'.

[1333] The definition, examples and preferred examples of the residue obtained by removing one hydrogen atom from Y in the above formula (B-1) are the same as those described above.

[1334] Therefore, the above formula (V1) can be defined as the following formula (V1');

[1335] [Chemical Formula 47]

[1336]

[1337] Where,

[1338] F, C1, p, q, X, R 1a 、R 1b , Z, R' and T's definitions, examples and preferred examples are the same as above,

[1339] Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1).

[1340] In a specific embodiment, in the above formula (V2), the structural unit represented by F-L1'-B can be represented by the following formula (FL1'B);

[1341] [Chemical Formula 48]

[1342]

[1343] Where,

[1344] F, p, q, X, R 1a 、R 1b , the definitions, examples and preferred examples of Y and Z are the same as those described above,

[1345] C1' is a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1346] ●(black circle) represents the bond to R'.

[1347] C1' represents a moiety generated by the reaction between a functional substance and a bioorthogonal functional group. The definition, examples, and preferred embodiments of the moiety generated by the reaction between a functional substance and a bioorthogonal functional group in C1' are the same as those described above in the "divalent group comprising a moiety generated by the reaction between a functional substance and a bioorthogonal functional group" (the same applies hereinafter).

[1348] Therefore, the above formula (V2) can be defined as the following formula (V2');

[1349] [Chemical Formula 49]

[1350]

[1351] Where,

[1352] F, p, q, X, R 1a 、R 1b The definitions, examples and preferred examples of , Y, Z, R' and T are the same as those described above,

[1353] C1' is a moiety generated by the reaction between the functional substance and the bioorthogonal functional group.

[1354] In a specific embodiment, in the above formula (V3), the structural unit represented by Fa-L1'-B'(-Fb) can be represented by the following formula (FaL1'B'(Fb));

[1355] [Chemical Formula 50]

[1356]

[1357] Where,

[1358] Fa, Fb, p, q, X, R 1a 、R 1b The definitions, examples and preferred examples of Z are the same as those described above,

[1359] C1' is a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1360] Y' is a residue obtained by removing one hydrogen atom from Y of the formula (B-1),

[1361] ●(black circle) represents the bond to R'.

[1362] Therefore, the above formula (V3) can be defined as the following formula (V3');

[1363] [Chemical Formula 51]

[1364]

[1365] Where,

[1366] Fa, Fb, p, q, X, R 1a 、R 1b , Z, R' and T's definitions, examples and preferred examples are the same as above,

[1367] C1' is a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[1368] Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1).

[1369] In the above formulas (L1B'(F)), (FL1'B) and (FaL1'B'(F2)), as well as (V1'), (V2') and (V3'), the length from C to C1 in C=Z is the same as the length of the partial structure connecting the L1 terminal part (or C1 terminal part) and R' generated by cleavage. In these formulas, the length from C to C1 in C=Z can also be specified according to the number of atoms constituting the connecting chain (excluding hydrogen atoms and substituents) constituting the partial structure from C to C1 in C=Z. Such number of atoms is the same as the number of atoms constituting the partial structure connecting the L1 terminal part (or C1 terminal part) and R' generated by cleavage. The connecting chain (excluding hydrogen atoms and substituents) connecting the partial structure from C to C1 in C=Z may contain a ring structure or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide part.

[1370] 5-3. Binding Sites of Antibodies Other Than Antibodies (Positional Selectivity)

[1371] The partial structure other than the antibody (e.g., F-(L1-B)'-R') can positionally selectively bind to the target region described above in the antibody (T). Specifically, when T contains one or more specific amino acid residues in a target region formed by 1 to 50 consecutive amino acid residues and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The definitions, examples, and preferred embodiments of the target region and positional selectivity are as described above.

[1372] 5-4. Number of partial structures other than antibodies possessed by an antibody

[1373] The number of partial structures other than the antibody (e.g., F-(L1-B)'-R') possessed by the antibody (T) may vary. For example, when T is a multimeric protein comprising a plurality of monomeric proteins, T may have partial structures other than T in a plurality of corresponding target regions in the plurality of monomeric proteins. Thus, T may have a plurality of partial structures other than T. Therefore, the structure represented by formula (V), (V1), (V2), (V3), (V1'), (V2') or (V3') indicates that T may have one or more partial structures other than T. The number of partial structures other than T possessed by T can be adjusted by appropriately setting conditions such as the type of antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number also varies depending on the type of antibody and may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[1374] In a specific embodiment, the number of partial structures other than antibodies possessed by an antibody can be multiple if the antibody is a multimeric protein composed of multiple monomeric proteins. Using the present invention, multiple partial structures other than antibodies can be introduced into the same target region of multiple monomeric proteins.

[1375] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred embodiments of antibodies are as described above. In the present invention, the number of partial structures other than antibodies retained by an antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[1376] 5-5. Manufacturing Method

[1377] The present invention provides a method for producing an antibody or a salt thereof having a functional substance. The production method can be classified into: (D) a method using a complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody or a salt thereof as a raw material (see Figure 2reaction (3)); and (E) a method using an antibody or a salt thereof having a bioorthogonal functional group as a raw material (refer to Figure 2 reaction (5)).

[1378] (D) A method using a complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof as a raw material, comprising the following steps:

[1379] (D1) cleaving the complex of the substance having affinity for the antibody, the cleavable moiety, the functional substance, and the antibody or a salt thereof by cleaving the cleavable moiety to generate the antibody or a salt thereof having the functional substance.

[1380] The complex comprising an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody is represented by formula (III), preferably formula (III'), and more preferably formula (III"). The antibody having a functional substance is represented by formula (V), preferably represented by formula (V1), (V2), or (V3), and more preferably represented by formula (V1'), (V2'), or (V3').

[1381] The "complex of a substance having affinity for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof" has a cleavable moiety that can be cleaved by the cleavage treatment described above and can therefore be cleaved. This cleavage reaction can be carried out as described in "4-6. Production Method" above.

[1382] The formation of an antibody or a salt thereof having a functional substance can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although this depends on the specific starting materials and the molecular weight of the product. Confirmation of positional selectivity, confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described in "2-6. Production Methods" above.

[1383] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising the following steps:

[1384] (D2) reacting an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety with a functional substance to generate a complex comprising the affinity substance for the antibody, the cleavage moiety, the functional substance, and the antibody, or a salt thereof; and

[1385] (D3) cleaving the complex of the substance having affinity for the antibody, the cleavable moiety, the functional substance, and the antibody or a salt thereof by cleaving the cleavable moiety to generate the antibody or a salt thereof having the functional substance.

[1386] The above-mentioned process (D2) can be carried out in the same manner as the above-mentioned process (B1) described in the above-mentioned "3-7. Manufacturing method". The above-mentioned process (D3) can be carried out in the same manner as the above-mentioned process (D1). The above-mentioned processes (D2) and (D3) can be carried out separately. Alternatively, the above-mentioned processes (D2) and (D3) can be carried out simultaneously depending on factors such as the combination of the cleavage portion, the functional substance and the bioorthogonal functional group. That is, when the above-mentioned processes (D2) and (D3) are carried out simultaneously, the product obtained in the above-mentioned process (D2) can be provided to the above-mentioned process (D3) without separating the product obtained in the above-mentioned process (D2).

[1387] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising the following steps:

[1388] (D4) reacting a compound comprising an affinity substance for the antibody, a cleavable moiety, and a reactive group, or a salt thereof, with the antibody to produce the antibody comprising the affinity substance for the antibody and the cleavable moiety, or a salt thereof;

[1389] (D5) reacting an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavage moiety with a functional substance to generate a complex comprising the affinity substance for the antibody, the cleavage moiety, the functional substance, and the antibody, or a salt thereof; and

[1390] (D6) cleaving the complex of the substance having affinity for the antibody, the cleavable moiety, the functional substance, and the antibody or a salt thereof by cleaving the cleavable moiety to generate the antibody or a salt thereof having the functional substance.

[1391] The above-mentioned step (D4) can be carried out in the same manner as the above-mentioned step (A1) described in the above-mentioned "2-6. Production method". The above-mentioned steps (D5) and (D6) can be carried out in the same manner as the above-mentioned steps (D2) and (D3). The above-mentioned step (D5) can be carried out in the same manner as the above-mentioned step (B1) described in the above-mentioned "3-7. Production method". The above-mentioned step (D6) can be carried out in the same manner as the above-mentioned step (D1). The above-mentioned steps (D4) to (D6) can be carried out separately. Alternatively, at least a part of the above-mentioned steps (D4) to (D6) can be carried out simultaneously depending on factors such as the combination of reactive groups, cleavable parts, functional substances and bioorthogonal functional groups. That is, when two or more of the above-mentioned steps (D4) to (D6) are carried out simultaneously, the product obtained in the previous step can be supplied to the next step without isolating the product obtained in the previous step.

[1392] (E) A method using an antibody or a salt thereof having a bioorthogonal functional group as a raw material, comprising the following steps:

[1393] (E1) reacting an antibody or a salt thereof having a bioorthogonal functional group with a functional substance to produce an antibody or a salt thereof having the functional substance.

[1394] The antibody having a bioorthogonal functional group is represented by Formula (IV), preferably Formula (IV'). The antibody having a functional substance is represented by Formula (V), preferably Formula (V1), (V2) or (V3), and more preferably Formula (V1'), (V2') or (V3').

[1395] Antibodies or salts thereof having bioorthogonal functional groups can react with functional substances because they have bioorthogonal functional groups. Such reactions can be carried out in the manner described in "3-7. Production Methods" above.

[1396] The production of an antibody or a salt thereof having a functional substance can be confirmed by the above-described method. The positional selectivity, the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described in "2-6. Production Method" above.

[1397] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising the following steps:

[1398] (E2) cleaving the cleavage portion of the antibody or its salt comprising an affinity substance for the antibody and a cleavage portion to generate an antibody or its salt having a bioorthogonal functional group; and

[1399] (E3) reacting the antibody or salt thereof having a bioorthogonal functional group with a functional substance to produce the antibody or salt thereof having the functional substance.

[1400] The above-mentioned process (E2) can be carried out in the same manner as the above-mentioned process (C1) described in the above-mentioned "4-6. Manufacturing method". The above-mentioned process (E3) can be carried out in the same manner as the above-mentioned process (E1). The above-mentioned processes (E2) and (E3) can be carried out separately. Alternatively, the above-mentioned processes (E2) and (E3) can be carried out simultaneously depending on factors such as the combination of the cleavage portion, the functional substance and the bioorthogonal functional group. That is, when the above-mentioned processes (E2) and (E3) are carried out simultaneously, the product obtained in the above-mentioned process (E2) can be provided to the above-mentioned process (E3) without separating the product obtained in the above-mentioned process (E2).

[1401] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising the following steps:

[1402] (E4) reacting a compound comprising an affinity substance for the antibody, a cleavable moiety, and a reactive group, or a salt thereof, with the antibody to produce the antibody comprising the affinity substance for the antibody and the cleavable moiety, or a salt thereof;

[1403] (E5) cleaving the cleavage portion of the antibody or its salt comprising an affinity substance for the antibody and a cleavage portion to generate an antibody or its salt having a bioorthogonal functional group; and

[1404] (E6) reacting the antibody or salt thereof having a bioorthogonal functional group with a functional substance to produce the antibody or salt thereof having the functional substance.

[1405] The above-mentioned process (E4) can be carried out in the same manner as the above-mentioned process (A1) described in the above-mentioned "2-6. Manufacturing method". The above-mentioned processes (E5) and (E6) can be carried out in the same manner as the above-mentioned processes (E2) and (E3). The above-mentioned processes (E4) to (E6) can be carried out separately. Alternatively, at least a part of the above-mentioned processes (E4) to (E6) can be carried out simultaneously depending on factors such as the combination of reactive groups, cleavable parts, functional substances and bioorthogonal functional groups. That is, when two or more of the above-mentioned processes (E4) to (E6) are carried out simultaneously, the product obtained in the previous process can be supplied to the next process without separating the product obtained in the previous process.

[1406] 6. Salt

[1407] In the present invention, examples of salts include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and magnesium), other metals such as zinc and aluminum, and ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). Salts with inorganic acids (e.g., hydrochloric acid) or organic acids (e.g., trifluoroacetic acid) are preferred.

[1408] 7. Purpose

[1409] The compounds of the present invention or their salts, which have an affinity substance for antibodies, a cleavable moiety, and a reactive group, can be used, for example, for position-selective modification of antibodies. Therefore, the present invention provides reagents for position-selective modification of antibodies, comprising a compound or its salt having an affinity substance for antibodies, a cleavable moiety, and a reactive group.

[1410] The position selective modification reagent of the present invention can be provided in the form of a composition further comprising other components. As such other components, for example, solutions, stabilizers (for example, antioxidants, preservatives (preservatives)) can be mentioned. As a solution, an aqueous solution is preferred. As an aqueous solution, for example, water (for example, distilled water, sterile distilled water, purified water, physiological saline), a buffer solution (for example, a phosphoric acid aqueous solution, a Tris-hydrochloric acid buffer solution, a carbonate-bicarbonate buffer solution, a boric acid aqueous solution, a glycine-sodium hydroxide buffer solution, a citrate buffer solution) can be mentioned, preferably a buffer solution. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The position selective modification reagent of the present invention can be provided in liquid or powder form (for example, a freeze-dried powder).

[1411] The antibody or salt thereof of the present invention (position-selectively) having an affinity substance for the antibody and a cleavage portion, the complex or salt thereof of the present invention (position-selectively) having an affinity substance for the antibody, a cleavage portion, a functional substance and an antibody, and the antibody or salt thereof of the present invention (position-selectively) having a bioorthogonal functional group can be used as an intermediate for preparing an antibody or a salt thereof (position-selectively) having a functional substance.

[1412] The antibody of the present in...

Claims

1. A compound or a salt thereof having an affinity substance for an antibody, a cleavable portion and a reactive group, wherein: The compound is represented by the following formula (I): ALBR(I) In the formula, A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R is an N-hydroxysuccinimide residue, The partial structure represented by LB does not contain the peptide part; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

2. The compound or salt thereof according to claim 1, wherein Glutamine residues are pyroglutamylated.

3. Use of a compound represented by the following formula (I) having an affinity substance for an antibody, a cleavable portion and a reactive group or a salt thereof in the preparation of a reagent for position-selective modification of an antibody, ALBR(I) In the formula, A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R is an N-hydroxysuccinimide residue, The partial structure represented by LB does not contain the peptide part; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

4. The use according to claim 3, wherein Glutamine residues are pyroglutamylated.

5. An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, wherein: The antibody is represented by the following formula (II): ALB-R'-T(II) In the formula, A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R' is an amide residue generated by the reaction between an antibody lysine residue and an N-hydroxysuccinimide residue, T is an antibody, The partial structure represented by LB does not contain the peptide part; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

6. The antibody or salt thereof according to claim 5, wherein The antibody in formula (II) has a plurality of structural units represented by ALB-R'.

7. The antibody or salt thereof according to claim 5 or 6, wherein Glutamine residues are pyroglutamylated.

8. A complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody or a salt thereof, wherein: The complex is represented by the following formula (III): AL-B'(-F)-R'-T(III) In the formula, A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B' is a trivalent group including a portion generated by a reaction between a functional substance and a bioorthogonal functional group, F is a functional substance, R' is an amide residue generated by the reaction between an antibody lysine residue and an N-hydroxysuccinimide residue, T is an antibody, The partial structure represented by L-B' does not contain a peptide portion; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

9. The complex or a salt thereof according to claim 8, wherein The antibody in formula (III) has a plurality of structural units represented by AL-B'(-F)-R'.

10. The complex or salt thereof according to claim 8 or 9, wherein Glutamine residues are pyroglutamylated.

11. A method for producing an antibody or a salt thereof having a bioorthogonal functional group, wherein: include: The cleavage portion of an antibody or a salt thereof having an affinity substance for the antibody and a cleavage portion represented by the following formula (II) is cleaved to generate an antibody or a salt thereof having a bioorthogonal functional group represented by the following formula (IV), The formula (II) is: ALB-R'-T(II) In formula (II), A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R' is an amide residue generated by the reaction between an antibody lysine residue and an N-hydroxysuccinimide residue, T is an antibody, The partial structure represented by LB does not contain the peptide part; The formula (IV) is: L1-B-R'-T(IV) In formula (IV), B, R' and T have the same meanings as the corresponding symbols in the formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

12. The method according to claim 11, wherein: The antibody in formula (II) has a plurality of structural units represented by ALB-R', and The antibody in formula (IV) has a plurality of structural units represented by L1-B-R'.

13. The method according to claim 11 or 12, wherein: Glutamine residues are pyroglutamylated.

14. A method for producing an antibody or a salt thereof having a functional substance, wherein: include: The complex of a substance having affinity for an antibody, a cleavable portion, a functional substance and an antibody represented by the following formula (III) or a cleavable portion of a salt thereof is cleaved to generate an antibody having a functional substance represented by the following formula (V1) or a salt thereof, wherein the formula (III) is: AL-B'(-F)-R'-T(III) In formula (III), A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B' is a trivalent group including a portion generated by a reaction between a functional substance and a bioorthogonal functional group, F is a functional substance, R' is an amide residue generated by the reaction between an antibody lysine residue and an N-hydroxysuccinimide residue, T is an antibody, The partial structure represented by L-B' does not contain a peptide portion; The formula (V1) is: L1-B'(-F)-R'-T(V1) In formula (V1), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group, and B', F, R' and T have the same meanings as the corresponding symbols in formula (III); Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and its amino acid sequence is selected from the following amino acid sequences: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

15. The method according to claim 14, wherein: The antibody in formula (III) has a plurality of structural units represented by AL-B'(-F)-R', and The antibody in formula (V1) has a plurality of structural units represented by L1-B'(-F)-R'.

16. The method according to claim 14 or 15, wherein: Glutamine residues are pyroglutamylated.

17. A method for producing an antibody or a salt thereof having a functional substance, wherein: include: (A) cleaving the cleavable portion of an antibody or a salt thereof having an affinity substance for the antibody and a cleavable portion represented by the following formula (II) to generate an antibody or a salt thereof having a bioorthogonal functional group represented by the following formula (IV); as well as (B) reacting the antibody or salt thereof having a bioorthogonal functional group with one or more functional substances to generate an antibody or salt thereof having a functional substance represented by the following formula (V2) or the following formula (V3), The formula (II) is: ALB-R'-T(II) In formula (II), A is an affinity substance for antibodies, L is a cleavable linker, which is a divalent group containing a disulfide residue, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group, R' is an amide residue generated by the reaction between an antibody lysine residue and an N-hydroxysuccinimide residue, T is an antibody, The partial structure represented by LB does not contain the peptide part; The formula (IV) is: L1-B-R'-T(IV) In formula (IV), B, R' and T have the same meanings as the corresponding symbols in the formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; The formula (V2) is: F-L1'-B-R'-T(V2) In formula (V2), B, R' and T have the same meanings as the corresponding symbols in formula (IV), L1' is a divalent group including a portion generated by a reaction between a functional substance and (i') a monovalent group including a bioorthogonal functional group, F is a functional substance; The formula (V3) is: Fa-L1'-B'(-Fb)-R'-T(V3) In formula (V3), R' and T have the same meanings as the corresponding symbols in formula (IV), L1' has the same meaning as the corresponding symbols in formula (V2), B' is a trivalent group including a portion generated by a reaction between a functional substance and a bioorthogonal functional group, Fa and Fb are respectively the same or different functional substances; Furthermore, the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus, and the amino acid residue is selected from the following amino acid sequence: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQA PQA (SEQ ID NO:8); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 25).

18. The method according to claim 17, wherein: The antibody in formula (II) has a plurality of structural units represented by ALB-R', The antibody in formula (IV) has a plurality of structural units represented by L1-B-R', The antibody in formula (V2) has a plurality of structural units represented by F-L1'-B-R', The antibody in formula (V3) has a plurality of structural units represented by Fa-L1'-B'(-Fb)-R'.

19. The method according to claim 17 or 18, wherein: Glutamine residues are pyroglutamylated.

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